Assembly of functionally integrated human forebrain spheroids and methods of use thereof

ABSTRACT

Human pluripotent stem cells are differentiated in vitro into forebrain subdomain structures, which are then fused to generate an integrated system for use in analysis, screening programs, and the like.

CROSS REFERENCE

This application claims benefit of U.S. Provisional Patent ApplicationNo. 62/477,858, filed Mar. 28, 2017, which application is incorporatedherein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract MH107800awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Progress in understanding the intricate development of the human centralnervous system and elucidating the mechanisms of neuropsychiatricdisorders in patients has been greatly limited by restricted access tofunctional human brain tissue. While studies in rodents and othermammals have provided important insights into the fundamental principlesof neural development, we know little about the cellular and molecularprocesses responsible for the massive expansion of the forebrain inprimates, nor many of its human specific features. In recent years, aparadigm shift has been achieved in the field with the introduction ofcellular reprogramming—a process during which terminally differentiatedsomatic cells can be converted into pluripotent stem cells, named humaninduced pluripotent stem cells (hiPSC). These hiPSCs can be generatedfrom any individual and, importantly, can be directed to differentiatein vitro into all germ layer derivatives, including neural cells.

While the methods and efficiency of generating hiPSCs have beensignificantly improved and standardized across laboratories, the methodsfor deriving specific neuronal cell types and glial cells remainchallenging. Over the past decade, improvements in neural specificationand differentiation protocols of pluripotent stem cells in monolayerhave led to the generation of a variety of cell types. Nonetheless,two-dimensional (2D) methods are unlikely to recapitulate thecyto-architecture of the developing three-dimensional (3D) nervoussystem or the complexity and functionality of in vivo neural networksand circuits. Moreover, these methods are laborious, costly, havelimited efficiency, give rise to relatively immature neurons andincompatible with long-term culturing of neurons.

Pharmaceutical drug discovery utilizes the identification and validationof therapeutic targets, as well as the identification and optimizationof lead compounds. The explosion in numbers of potential new targets andchemical entities resulting from genomics and combinatorial chemistryapproaches over the past few years has placed massive pressure onscreening programs. The rewards for identification of a useful drug areenormous, but the percentages of hits from any screening program aregenerally very low. Desirable compound screening methods solve thisproblem by both allowing for a high-throughput so that many individualcompounds can be tested; and by providing biologically relevantinformation so that there is a good correlation between the informationgenerated by the screening assay and the pharmaceutical effectiveness ofthe compound.

Some of the more important features for pharmaceutical effectiveness arespecificity for the targeted cell or disease, a lack of toxicity atrelevant dosages, and specific activity of the compound against itsmolecular target. Therefore, one would like to have a method forscreening compounds or libraries of compounds that allows simultaneousevaluation for the effect of a compound on the biologically relevantcell population, where the assay predicts clinical effectiveness.

The effect of drugs on specific human neural or glial cell types is ofparticular interest, where efficacy and toxicity may rest insophisticated analysis of cell migration, activity, or the ability ofneurons to form functional networks, rather than on simple viabilityassays. The discrepancy between the number of lead compounds in clinicaldevelopment and approved drugs may partially be a result of the methodsused to generate the leads and highlights the need for new technology toobtain more detailed and physiologically relevant information oncellular processes in normal and diseased states.

A number of important clinical conditions are associated with alteredneuronal or glial function, including neurodegenerative disorders(Alzheimer's disease (AD), Parkinson's disease (PD), Huntington'sdisease (HD), and amyotrophic lateral sclerosis (ALS), or psychiatricconditions such as schizophrenia and other psychoses, bipolar disorders,mood disorders, intellectual disability (ID) or autism spectrumdisorders (ASD).

In addition to pharmaceutical drug discovery, there is a pressing needfor meaningful screening platforms to identify and explore specifictoxicity effects due to the increasing number of new therapeuticcompounds and chemical substances with human exposure. Particularly, inthe field of neurotoxicity, assays capable of assessing the impairmentof neuronal or glial function are still lacking for human cells.

Therefore, the development of in vitro screening platforms thatrecapitulate highly functional human tissue is of utmost importance.

Publications. Methods to reprogram primate somatic cells to apluripotent state include differentiated somatic cell nuclear transfer,differentiated somatic cell fusion with pluripotent stem cells anddirect reprogramming to produce induced pluripotent stem cells (iPScells) (Takahashi K, et al. (2007) Cell 131:861-872; Park I H, et al.(2008) Nature 451:141-146; Yu J, et al. (2007) Science 318:1917-1920;Kim D, et al. (2009) Cell Stem Cell 4:472-476; Soldner F, et al. (2009)Cell. 136:964-977; Huangfu D, et al. (2008) Nature Biotechnology26:1269-1275; Li W, et al. (2009) Cell Stem Cell 4:16-19).

SUMMARY OF THE INVENTION

Compositions and methods are provided for in vitro generation of a human3D microphysiological system (MPS) that comprisesfunctionally-integrated excitatory glutamatergic and GABAergic neurons.This system is generated by the directed differentiation of subdomainsof the forebrain that functionally interact in development. Subdomainsare then assembled (fused) into a functionally integrated forebrainspheroid. The system captures in vitro processes typical of in vivocentral nervous system (CNS) development that could not otherwise bemodeled with conventional 2D cultures, including the saltatory migrationof interneurons on their way to the cerebral cortex. After migratinginto an active neural network, interneurons mature and integrate into asynaptically connected microphysiological system without the requirementof seeding onto rodent cortical cultures or brain slice cultures.Assembling networks using this modular system allows the study ofexcitation to inhibition interplay during cortical development in normaland disease states. More importantly, this system illustrates a novelconcept for deriving and then assembling developmentally and diseaserelevant human brain regions from pluripotent stem cells (hESC, hiPSC)with the goal of capturing novel, emerging features of the CNS and toenable mechanistic and therapeutic studies of these processes in vitro.For instance, one could assemble human cortico-cortical spheroids tomimic interhemispheric axonal guidance and communication, but alsocorticostriatal spheroids, striato-nigral, cortico-spinal, etc.

In one embodiment, methods are provided for the generation of a human 3Dmicrophysiological (MPS) forebrain system. The methods comprise aninitial step of differentiating pluripotent cells (hPSC) into theforebrain subdomains of (i) a ventral forebrain structure, referred toherein as a subpallial spheroid (hSS) comprising primarily GABAergiccortical interneurons; and (ii) a cerebral cortical, or dorsal palliumstructure (hCS) comprising cortical gluamatergic neurons of variouslayers. These neural spheroids may also comprise neural progenitorcells, astrocytes, oligodendrocytes, neurons and the like. Followingthis differentiation step, subpallial spheroid(s) (hSS) and corticalspheroid(s) (hCS) are placed adjacent to each other in culture underconditions permissive for fusion of the two spheroids and generation ofthe integrated forebrain system. One or both of the spheroids maycomprise cells detectably labeled with, for example, a fluorescent orluminescent expressed protein marker. During the fusion process, thesaltatory migration of neurons from hSS to hCS can be observed. Thefused forebrain comprises functionally integrated cortical neurons ofexcitatory and inhibitory types, which provides a platform for analysisof the effect of agents on brain structure and function. The validity ofthis platform has been assessed by performed live imaging ofDlxi1/2b::eGFP-labeled cells in human forebrain tissue at gestationalweeks 18 and 20. More specifically, it was observed that the averagesaltation length in fetal forebrain tissue was 43.54 μm±2.39 and verysimilar to the 38.17 μm±1.33 saltation length observed in fused hSS-hCS.Similarly, the average frequency of saltation per 8 hours was 3.08±0.18in human fetal tissue versus 2.9±0.25 saltations in fused hSS-hCS. Takentogether, these data suggests that this platform recapitulates withgreat accuracy the migration pattern of human cortical interneurons inthe developing forebrain. Live imaging data and 3D reconstructions withoptical clearing methods of fused (assembled) hSS-hCS indicate thatinterneurons migrating close to the surface of hCS and often encounterlayer 1 neurons (RELN⁺, TBR1⁺), which are positioned on the surface ofhCS, reminiscent of a marginal zone-like migration. At the same time,interneurons at the hSS-hCS interface often penetrate and encountercortical glutamatergic neurons, which are primarily deep-layer corticalneurons at this in vitro developmental stage of hCS. This deepermigration also makes possible for some of these interneurons to getcloser to pallial proliferative zones and undergo ventricular-directedmigration.

In some embodiments, methods are provided for determining the activityof a candidate agent on human cells present in the integrated forebrainsystem or isolated from the integrated forebrain system, the methodcomprising contacting the candidate agent with one or a panel ofintegrated forebrain system or purified cells derived therefrom. Thecell populations optionally comprise at least one allele encoding amutation associated or potentially with a neuropsychiatric disease; anddetermining the effect of the agent on morphological, genetic orfunctional parameters, including without limitation gene expressionprofiling. Methods of analysis at the single cell level are also ofinterest, e.g. migration assays, axonal growth and pathfinding assays,atomic force microscopy, super resolution microcopy, light-sheetmicroscopy, two-photon microscopy, patch clamping, single cell geneexpression (RNA-seq), calcium imaging with pharmacological screens,modulation of synaptogenesis, and the like.

In some embodiments, one or more such integrated forebrain systems areprovided, including without limitation a panel of such in vitro derivedintegrated forebrain systems are provided, where the panel includes twoor more genetically different cells. In some embodiments a panel of suchintegrated forebrain systems are provided, where the systems can besubjected to a plurality of candidate agents, or a plurality of doses ofa candidate agent. Candidate agents include small molecules, i.e. drugs,genetic constructs that increase or decrease expression of an RNA ofinterest, infectious agents, electrical changes, and the like. In someembodiments a panel refers to a system or method utilizingpatient-specific systems from two or more distinct conditions, and maybe three or more, four or more, five or more, six or more, seven or moregenetically distinct conditions.

In one embodiment, methods are provided for generating human subpallialspheroids (hSS) and cells comprised therein, including, for exampleneural progenitors and GABAergic interneurons. A feature of theinvention is the ability to generate these cells, and systems comprisingsuch cells, from patient samples, allowing disease-relevant generationand screening of the cells for therapeutic drugs and treatment regimens,where the methods utilize in vitro cell cultures or animal modelsderived therefrom for such purposes. The methods utilize induced humanpluripotent stem cells (hiPSCs), which may be obtained from patient orcarrier cell samples, e.g. adipocytes, fibroblasts, keratinocytes, bloodcells and the like. The hiPSCs are instructed to develop a neural fatein vitro, and then specified into human subpallial spheroids (hSS). Thecell populations can be isolated from the hSS, the intact hSS can beused as a model for interacting neural cell populations, or fused to anhCS as a model for more complex neural interactions. The hSS and cellsderived therefrom may be used for transplantation in mammals and otherspecies, for experimental evaluation including screening of drugs andbiological entities, as a source of lineage and cell specific products,and the like. In some embodiments the cell cultures are feeder-free andxeno-free. In some embodiments of the invention, populations of purifiedhuman cells from the spheroids (hCS, hSS), e.g. GABAergic interneuronsand progenitors thereof, etc. are provided, where the cells aredifferentiated from induced human pluripotent stem cells (hiPSCs). Insome embodiments the spheroids find use in analyzing cell-autonomous orregion-autonomous phenotypes in disease states. Various combinations ofassemblies can be generated, for example a cortical spheroid generatedfrom control-derived (normal) hiPSCs can be assembled with a subpalliumspheroids generated from patient-derived (disease state) hiPSCs and thelike.

After differentiation in hSSs or integrated forebrain system, individualcell types of interest can be isolated for various purposes. The cellsare harvested at an appropriate stage of development, which may bedetermined based on the expression of markers and phenotypiccharacteristics of the desired cell type. Cultures may be empiricallytested by immunostaining for the presence of the markers of interest, bymorphological determination, etc. The cells are optionally enrichedbefore or after the positive selection step by drug selection, panning,density gradient centrifugation, flow cytometry etc. In anotherembodiment, a negative selection is performed, where the selection isbased on expression of one or more of markers found on hESCs,fibroblasts, neural cells, epithelial cells, and the like. Selection mayutilize panning methods, magnetic particle selection, particle sorterselection, fluorescent activated cell sorting (FACS) and the like.

Various somatic cells find use as a source of hiPSCs; of particularinterest are adipose-derived stem cells, fibroblasts, and the like. Theuse of hiPSCs from individuals of varying genotypes, particularlygenotypes potentially associated with neurologic and psychiatricdisorders or even idiopathic forms of neuropsychiatric disease (autismspectrum disorders, psychosis, etc) are of particular interest. ThehiPSCs are dissociated and grown in suspension; then induced to a neuralfate by inhibition of BMP and ROCK pathways. The spheroids are thenmoved to neural medium in the presence of FGF2 and EGF. To generate thehSS, the medium further comprises modulators of Wnt and sonic hedgehog(SHH) signaling pathways, and may optionally also comprise one or bothof allopregnanolone and transient exposure to retinoic acid. Thespheroids are then changed to medium comprising the growth factors BDNFand NT3. After such culture, the spheroids can be maintained forextended periods of time in neural medium in the absence of growthfactors, e.g. for periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24,36 months or longer.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the subject methods and compositions as more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1N. Characterization of hSS derived from hiPSC. (FIG. 1A) Schemeillustrating the main stages for the generation of hCS and hSS fromhiPSC. (FIG. 1B) Fold changes in gene expression (relative to expressionin hiPSC and normalized to GAPDH) of NKX2-1 (n=6 hiPSC lines;Mann-Whitney test, P=0.002), FOXG1 (n=5 hiPSC lines; t-test, P=0.35) andEMX1 (n=4 hiPSC lines; Mann-Whitney test, P=0.02) in hCS and hSS at day25. (FIG. 1C, 1D) Immunostaining for NKX2-1 in cryosections of hSS atday 25 and day 60 of differentiation. (FIG. 1E, 1F) Immunostaining incryosections of hSS showing expression of GABA and GAD67 and theneuronal marker MAP2 (day 60 of differentiation). (FIG. 1G, 1H) Examplesof immunostaining showing cells expressing the GABAergic subtype markerssomatostatin (SST), calretinin (CR), calbindin (CB) at day 60 and day109, and parvalbumin (PV) at day 209. (FIG. 1I) t-SNE visualization ofsingle cell gene expression in hCS (magenta) and hSS (green) at day 105of differentiation (n=11,838 cells; BD™ Resolve system). (FIG. 1J, 1K)Main single cell clusters and boxplots for genes enriched in eachcluster (expression of the top 25 genes in each cluster is shown in FIG.6d-k . (FIG. 1L) Volume rendering by array tomography of the interior ofa hSS (19.4×18.4×2.9 μm) showing expression of the neuronal marker MAP2(red), the glial marker GFAP (cyan), and the synaptic proteins SYN1(green) and VGAT (magenta) (DAPI shown in white). (FIG. 1M) Schemeillustrating patch clamping in sliced hSS and a representative trace ofwhole-cell current-clamp recording. (FIG. 1N) Representative traces andquantification of spontaneous IPSCs recorded before (black) and during(blue) application of Gabazine (GBZ, 10 μM) in an acute slicepreparation of hSS (paired t-test, **P=0.004). Representative traces ofEPSCs recorded from hCS are shown in FIG. 9 a.

FIG. 2A-2P. Cell migration in fused hSS-hCS. (FIG. 2A) Schemeillustrating the assembly of hCS and hSS. Cells in hSS are labeled withDlxi1/2b::eGFP prior to fusion. (FIG. 2B) Morphology of hCS and hSSbefore and after assembly. (FIG. 2C) Time-lapse of migration ofrepresentative Dlxi1/2b::eGFP+ cells from hSS into hCS over 24 days.(FIG. 2D) Representative example showing the assembly of a fluorescentlylabeled hCS (AAV-hSyn1::mCherry) and a fluorescently labeled hSS(Lenti-Dlxi1/2b::eGFP) after 8 days. (FIG. 2E) 3D reconstruction of aniDISCO-cleared hSS-hCS at 24 days after fusion. (FIG. 2F) Representativeexample of time-lapse live imaging showing the saltatory migration ofDlxi1/2b::eGFP+ cells in fused hSS-hCS. Yellow arrowheads indicate somaand nucleokinesis. (FIG. 2G) Cumulative plot showing the saltatorymigration and pausing of representative Dlxi1/2b::eGFP+ cells in fusedhSS-hCS (n=5 cells). (FIG. 2H) Representative immunostaining showing aDlxi1/2b::eGFP+ cell undergoing nucleokinesis. Yellow arrowheadsindicate the cell soma. (FIG. 2I) Representative example of time-lapselive imaging showing the saltatory migration of Dlxi1/2b::eGFP+ cells inhuman fetal forebrain (GW18). Yellow arrowheads indicate soma andnucleokinesis. (FIG. 2J) Cumulative plot showing the saltatory migrationand pausing of representative Dlxi1/2b::eGFP+ cells in human fetalforebrain (n=5 cells). (FIG. 2K) Representative immunostaining showing aDlxi1/2b::eGFP+ cell undergoing nucleokinesis in slices of human fetalforebrain at GW18. Yellow arrowheads indicate the cell soma. (FIG. 2L)Scheme illustrating the pharmacological manipulation of Dlxi1/2b::eGFP+cells migrating in hSS-hCS. (FIG. 2M, 2N, 2O, 2P) Quantification ofDlxi1/2b::eGFP+ cell migration before and after exposure to 100 nM ofthe CXCR4 antagonist AMD3100 (n=8 cells from 2 hiPSC lines; pairedt-tests, *P=0.03 for number of saltations, **P=0.006 for saltationlength, **P=0.006 for speed when mobile, *P=0.02 for path directness).Tracking of the migration path of individual Dlxi1/2b::eGFP+ cells isshown in FIG. 10 t.

FIG. 3A-3I. Modeling of interneuron migration in hSS-hCS derived fromTimothy syndrome. (FIG. 3A) Scheme depicting the L-type calcium channel(LTCC) Cav1.2 and the p.G406R gain-of-function mutation (blue star) thatcauses TS. (FIG. 3B) Calcium imaging (Fura-2) in dissociated hSS derivedfrom TS subjects and controls (Ctrl: n=38 cells from 2 subjects; TS:n=68 cells from 2 subjects). (FIG. 3C) Quantification of residualintracellular calcium ([Ca2+]i) following depolarization of Ctrl and TScells in hSS. Residual intracellular calcium was calculated by dividingthe plateau calcium (C-A) level by the peak calcium elevation (B-A);(t-test, ***P<0.0001). Residual intracellular calcium in Ctrl and TS hCScells is shown in FIG. 13h . (FIG. 3D) Time-lapse of migration ofrepresentative Dlxi1/2b::eGFP+ cells in TS and control hSS-hCS. Yellowarrowheads indicate soma and nucleokinesis. A representative image offused hCS-hSS shown in FIG. 13i . (FIG. 3E, 3F) Quantification of thenumber of saltations (Ctrl: n=48 cells from 3 hiPSC lines from 3subjects; TS: n=51 cells from 3 hiPSC lines from 3 subjects; TS-Ctrlhybrid: n=24 cells from 5 hiPSC line combinations), and saltation length(Ctrl: n=21 cells from 3 hiPSC lines from 3 subjects; TS: n=29 cellsfrom 3 hiPSC from 3 subjects; TS-Ctrl hybrid: n=12 cells from 3 hiPSCline combinations); one-way ANOVA with Dunnett's multiple comparisontest (***, P<0.001). Averages for individual hiPSC lines are shown inFIG. 13j-l . (FIG. 3G) Cumulative plot showing migration of TS andcontrol Dlxi1/2b::eGFP+ cells in fused hSS-hCS (two-way ANOVA,interaction F(24, 408)=17.71, P<0.0001). (FIG. 3H) Scheme illustratingpharmacological manipulation of LTCC during live imaging of fusedhSS-hCS in TS versus Ctrl. (FIG. 3I) Quantification of saltation lengthfollowing exposure to the LTCC antagonist nimodipine (5 μM) in TS versuscontrol (paired t-tests; Ctrl: n=13 cells from 3 hiPSC lines from 3subjects, ***P<0.001; TS: n=12 cells from 2 hiPSC lines from 2 subjects,***P<0.001).

FIG. 4A-4M. Functional integration of interneurons in fused hSS-hCS.(FIG. 4A) Scheme showing the isolation by dissociation and FACS ofDlxi1/2b::eGFP+ cells from the hSS or hCS side of 4-week fused hCSS-hSSfor single cell transcriptional analysis. (FIG. 4B) t-SNE visualizationof single cell gene expression of Dlxi1/2b::eGFP+ cells isolated fromhSS and hCS of fused hSS-hCS at day 121 of differentiation (4 weeksafter forebrain spheroid assembly; n=181 cells; cells form 2 hiPSClines; Smart-seq2 system). (FIG. 4C) Distribution of cells acrossclusters A, B and C (χ²-test, χ²=43.39, P<0.0001). (FIG. 4D) Violinplots showing expression (normalized log 2 transformed) inDlxi1/2b::eGFP+ cells in clusters A, B, and C (likelihood ratio test;ERBB4, NXPH1, IGF1, TCF4: P<e⁻⁶ for B versus A & C; FOS, RAD1: P<e⁻⁸ forC versus A & B). (FIG. 4E) Representative morphologies ofDlxi1/2b::eGFP+ cells before and after migration into hCS in fusedhSS-hCS. A 3D reconstruction of Dlxi1/2b::eGFP+ cells before and aftermigration is shown in FIG. 14a . (FIG. 4F) Quantification of dendriticbranching of Dlxi1/2b::eGFP+ cells before (n=58 cells) and after (n=55cells) fusion of hSS to hCS (two-way ANOVA; interaction F(2, 129)=11.29,P<0.001; Bonferroni post-hoc *P<0.05, ***P<0.001). (FIG. 4G) Actionpotential generation (slice recordings) in Dlxi1/2b::eGFP+ cells inunfused hSS, in hSS of fused hSS-hCS and in hCS after migration in fusedhSS-hCS (one-way ANOVA, F(2, 30)=1.25; ***P<0.001; Bonferroni post-hoc,**P<0.01; ***P<0.001). Representative traces are shown in FIG. 14b .(FIG. 4H) Array tomography showing a volume reconstruction (4.0×4.0×2.1μm) on the pallial side of fused hCS-hSS with SYN1 (red) and theGABAergic synapse proteins GPHN (green) and VGAT (cyan). (FIG. 4I)Synaptogram of a Dlxi1/2b::eGFP+ cell illustrating the colocalizationwith SYN1 (red), GPHN (cyan), and VGAT (white); 5 consecutive 70 nmsections (3×3 μm). (FIG. 4J) Representative traces of EPSCs and IPSCs inDlxi1/2b::eGFP+ cells after migration into hCS. (FIG. 4K) Quantificationof synaptic responses in Dlxi1/2b::eGFP+ cells (IPSCs in green, EPSCs inmagenta) in hSS, in hSS of fused hSS-hCS and in hCS after migration infused hSS-hCS (two-way ANOVA, interaction F(2, 61)=18.46, P<0.0001;Bonferroni post-hoc for EPSCs, ***P<0.0001, **P<0.001). Representativetraces are shown in FIG. 14d . (FIG. 4L) Quantification of synapticresponses in excitatory cells (IPSCs in green, EPSCs in magenta) in hCS,in hCS of fused hSS-hCS and in hCS after migration in fused hSS-hCS(two-way ANOVA, cortical neurons in hCS before and after fusion F(1,26)=5.6, P<0.05; Bonferroni post-hoc for IPSC, *P<0.05). Representativetraces are shown in FIG. 14e (FIG. 4M) Electrical stimulation and patchclamp recording in fused hSS-hCS showing evoked EPSCs and IPSCs before(black trace) and after exposure to 10 μM Gabazine (red trace).Quantification of pre- and post-stimulus events is shown in FIG. 14 f.

FIG. 5. Immunostaining of hSS in cryosections showing Parvalbumin (PV)neurons. Two anti-PV antibodies have been used for validation ofspecificity; co-localization with the neuronal marker DCX (day 209).

FIG. 6A-6L. t-SNE visualization of single cell gene expression of hCSand hSS at day 105 of differentiation (n=11,838 cells; BD Resolvesystem) (FIG. 6A) Distribution of expression of the neuronal markerSTMN2, (FIG. 6B) the progenitor marker VIM and of (FIG. 6C) a set ofgenes associated with the M cell cycle phase (AURKB, TPX2, UBE2C, HMMR,TOP2A, CCNB1, NUSAP1, NUF2, CDC6, HIST1H4C, BIRC5, CKS2). (FIG. 6D-6K)top 25 genes in each of the 8 clusters shown in FIG. 1j (proportion ofmolecules per cells). (FIG. 6L) Scatter plot showing the number of genesdetected versus the number of useful reads.

FIG. 7A-7B. Calcium imaging of intact hSS (Fluo-4). (FIG. 7A)Representative traces of intracellular calcium measurements (Fluo-4)demonstrating spontaneous activity in hSS at ˜day 50 of differentiation.(FIG. 7B) Average calcium spike frequency across three distinct hSSdifferentiation conditions: IS (n=114 cells), ISA (n=327), ISRA (n=136);cells from 3 hiPSC lines; one-way ANOVA, P=0.006.

FIG. 8A-8J. Characterization of hSS differentiation conditions. (FIG.8A) Schematic illustrating the differentiation conditions for derivinghCS or hSS: IS, ISA and and ISRA. (FIG. 8B) Gene expression (RT-qPCR,normalized to GAPDH) showing down-regulation of OCT3/4 and the lack ofmesoderm (BRACH) and endoderm (SOX17) markers following differentiationof hiPSC into hCS and hSS conditions. (FIG. 8C) Gene expression (RT-PCR,fold change versus hiPSC and normalized to GAPDH) showing upregulationof forebrain markers (SIX3, FOXG1) but not midbrain (LMX1B),hypothalamus (RAX1) or spinal cord (HOXB4) markers. (FIG. 8D) Expressionof ventral forebrain genes in hSS and hCS (RT-qPCR, normalized to GAPDH)at day 25. (FIG. 8E) Average percentage of the proportion of NKX2-1 byimmunostaining in dissociated hCS or hSS at day 25. (FIG. 8F) Expressionof ventral forebrain genes in hSS (RT-qPCR, normalized to GAPDH) at day60. (FIG. 8G) Expression of glutamatergic, GABAergic, dopaminergic andcholinergic neurotransmitter identify genes in hSS (RT-qPCR, normalizedto GAPDH) at day 60. (FIG. 8H) Average percentage of the proportion ofMAP2/Hoechst and GAD67/MAP2 by immunostaining in dissociated hSS at day60. (FIG. 8I, 8J) Expression of interneuron subtypes genes and markersassociated with interneuron migration in hSS (RT-qPCR, normalized toGAPDH) at day 60. (n=3-6 hiPSC lines from 1-3 differentiations).

FIG. 9A-9B. (FIG. 9A) Representative EPSCs traces quantification ofrecordings from hCS neurons (sliced preparation) before (black trace)and during (green trace) exposure to the glutamate receptor blockerkynurenic acid (1 mM) (Mann-Whitney test, **P=0.007). (FIG. 9B) Overlapof averaged EPSCs (red trace) recorded in hCS neurons and averaged IPSCs(black trace) recorded in hSS (n=5-6; mean±standard deviation).

FIG. 10A-10T. Migration of Dlxi1/2::eGFP+ cells in fused hSS-hCS. (FIG.10A, 10B) Representative immunostaining in cryosections of hSS showingco-expression of Dlxi1/2::eGFP and GAD67 and GABA. (FIG. 10C)Quantification by immunostaining of the proportion of Dlxi1/2::eGFP+cells that co-express GAD67 in hSS derived using the ISA or ISRAcondition (n=3 lines; t-test, P=0.35). (FIG. 10D) Proportion ofDlxi1/2::eGFP+ cells in hSS derived using the ISA or ISRA condition thatco-express somatostatin (SST, t-test, P=0.48), calretinin (CR, t-test,*P=0.04) or calbindin by immunostaining (CB, t-test, P=0.43); n=3 linesfor SST, CR and CB quantifications. (FIG. 10E) Representative images offused spheroids (at day 60) showing migration of Dlxi1/2b::eGFP+ cells(from labeled hSS) in fused hSS-hCS but not in hSS-hSS over time. (FIG.10F) The number of Dlxi1/2b::eGFP+ (hSS-derived) or hSyn1::mCherry+cells (hCS-derived) that moved in fused hSS-hCS or hSS-hSS wasquantified in snapshots of live, intact spheroids at different timepoints (from day 3 to day 25). The values shown are absolute number ofcells that migrated to the other side (approximately the same area andthickness was imaged in each session) (one-way ANOVA for cells at 17days after fusion/assembly; F(2, 32)=8.24, P=0.001). (FIG. 10G)Representative images of fused spheroids (at day 91) showing migrationof Dlxi1/2b::eGFP+ cells (from labeled hSS) in fused hSS-hCS. (FIG. 10H)Representative image of an hSS that was plated on a coverslip andcultured for ˜7 days. (FIG. 10I) Percentage of Dlxi1/2::eGFP inside thecoverslip plated hSS and in the rim (0-200 μm) or beyond this region(>200 μm). (FIG. 10J) Quantification of the number of saltations ofDlxi1/2b::eGFP+ cells (n=32) inside the one-week plated hSS, in the rimand outside this region (one-way ANOVA, interaction F (2, 30)=22.12,P<0.001; Bonferroni post-hoc ***P<0.0001). (FIG. 10K) Diagram showingthe angle of movement of migrating Dlxi1/2b::eGFP+ cells at 8-9 daysafter fusion of hSS-hCS. The angle was calculated between the leadingprocess of Dlxi1/2b::eGFP+ cells that have moved into hCS and the fusioninterface (n=92 cells in 15 fused hSS-hCS from 5 hiPSC lines and 4independent differentiations). (FIG. 10L) Histogram showing thedistribution of the distance of migrated Dlxi1/2b::eGFP+ cells relativeto the edge of hCS in hSS-hCS at 30-50 days after fusion. The distancewas measured in fixed 18 μm cryosections (n=73 cells from 11 fusedhSS-hCS derived from 2 hiPSC lines in 2 independent differentiations).(FIG. 10M, 10N, 10O) Representative example of Dlxi1/2b::eGFP+ cellsmigrated in the hCS that moved within a ventricular zone-like (VZ)region. The VZ-like region shows GFAP-expressing cells, is surrounded byTBR1 expressing-cells and the migrated cells show GABA expression. (FIG.10P, 10Q, 10R, 10S) Representative images of immunostainings (SST,GAD67, GABA, CR, CB) of Dlxi1/2b::eGFP+ cells after migration in fusedhSS-hCS. (FIG. 10T) Plot illustrating the trajectory of Dlxi1/2b::eGFP+cells in fused hSS-hCS before and after exposure to the CXCR4 antagonistAMD3100.

FIG. 11A-11E. Single cell gene expression in Dlxi1/2b::eGFP+ cellsbefore and after migration (Smart-seq2). (FIG. 11A) Scheme showing theisolation by dissociation and FACS of Dlxi1/2b::eGFP+ cells from hSS orhCS for single cell transcriptional analysis. (FIG. 11B) Violin plotsshowing expression (normalized log 2 transformed) in Dlxi1/2b::eGFP+cells of selected genes associated with cortical, striatal and olfactoryinterneurons in hSS (light green, n=123 cells) or hCS (dark green; n=106cells) 2 weeks after assembly of hSS-hCS. (FIG. 11C) Violin plotsshowing expression (normalized log 2 transformed) in Dlxi1/2b::eGFP+cells (after 4 weeks of migration) in clusters A, B, and C (likelihoodratio test; GAD1, CELF4: P>0.05; PBX3: P<e⁻⁷ for A versus B & C;NNAT:P<e−16 for C versus A & B, P<e⁻¹⁶ for B versus A & C; MALAT1: P<e⁻⁹for C versus A & B; SOX11: P<e⁻¹⁶ for B versus A & C, P<e⁻⁹ for A versusB & C; GRIP2: P<e⁻⁸ for B versus A & C). (FIG. 11D) Scatter plot showingthe number of genes detected (≥10 reads cutoff) versus the number ofreads (n=410 cells from combined single cell RNA-seq experiments after 2weeks or 4 weeks of assembly in hSS-hCS). (FIG. 11E) Graph illustratingbiologically variable transcripts (red circles) and non-variabletranscripts (black circles) along with the technical noise from the ERCCspike in RNAs (blue dots). Green line shows the technical noise fit.

FIG. 12A-12L. Migration of Dlx2i1/2b::eGFP cells in mice brain slicesand hSS-hCS. (FIG. 12A) (FIG. 12B) (FIG. 12C) Representative images ofhuman fetal cortex at GW20 showing immunostaining with antibodiesagainst GFAP, CTIP2 and GABA. (FIG. 12D) Representative image showingcell labeling with the Dlx2i1/2b::eGFP reporter in fetal human forebrainat GW18 (6 days after lentivirus infection) (FIG. 12E, FIG. 12F)Representative immunostainings in cryosections of human forebrain tissueat GW18 showing co-localization of Dlx2i1/2b::eGFP with NKX2-1 and GABA.(FIG. 12G) Representative images showing cell labeling with theDlx2i1/2b::eGFP reporter in hSS-hCS (9 daf), in fetal human forebrain(GW18) and in mouse slice cultures (E18). (FIG. 12H, FIG. 12I)Comparison of Dlx2i1/2b::eGFP+ cell size and quantification of the ratioof soma diameter to the length of the leading process in fused hSS-hCS(n=25 cells from 4 hiPSC lines), human fetal forebrain at GW18 (n=19cells; black dots) and GW20 (n=15 cells; gray dots), hSS-derivedcultured on E14 mouse forebrain slices (n=14 cells), and E18 mouseforebrain slices (n=30 cells from 2 litters) (one-way ANOVA, interactionF(3, 97)=11.61, P=0.001, Bonferroni post-hoc ***P<0.001, **P<0.05).(FIG. 12J, 12K, 12L) Comparison of the number of saltations (one-wayANOVA, interaction F(2, 103)=29.27, P=0.001, Bonferroni post-hoc***P<0.001), saltation length (one-way ANOVA, interaction F(2, 91)=3.0,P=0.50), speed when mobile (one-way ANOVA, interaction F(2, 83)=11.38,P=0.001, Bonferroni post-hoc ***P<0.001) for Dlx2i1/2b::eGFP+ in fusedhSS-hCS (n=38-56 cells from 2-3 hiPSC lines), human fetal forebrain(GW18: n=19 cells; GW20: n=15 cells), and E18 mouse forebrain slices(n=14-16 cells from 2 litters).

FIG. 13A-13U. Derivation of TS hSS, migration and electroporation (FIG.13A) Sequencing of PCR-amplified DNA showing the p.G406R mutation inexon 8a of CACNA1C in TS (subject 8303). (FIG. 13B) Representativepictures of iPSC colonies expressing pluripotency markers (OCT4, SSEA4)in one TS subject (FIG. 13C) Level of expression (RT-qPCR, normalized toGAPDH) for NKX2-1 showing no major defects in ventral forebraininduction in TS (two-way ANOVA; interaction F(2,15)=0.20, P=0.81; TS vsCtrl F(1,15)=0.16, P=0.68). (FIG. 13D-13F) Representativeimmunostainings in cryosections of TS hSS (day 60 of differentiation)showing expression of NKX2-1, GABA, MAP2, GAD67, SST and CR. (FIG. 13G)Calcium imaging (Fura-2) in dissociated hCS derived from TS subjects andcontrols (Ctrl: n=81 cells from 2 subjects; TS: n=147 cells from 2subjects). (FIG. 13H, 13I) Quantification of residual intracellularcalcium ([Ca2+]i) following 67 mM KCl depolarization of Ctrl and TScells in hSS cells. Residual intracellular calcium was calculated bydividing the plateau calcium (C-A) level by the peak calcium elevation(B-A); (t-test, ***P<0.0001). (FIG. 13J) Representative image of fusedTS hSS-hCS showing Dlxi1/2b::eGFP expression and migration. (FIG. 13K,13L) Quantification of the number of saltations and saltation length ofDlx2i1/2b::eGFP cells in fused hSS-hCS across multiple Ctrl and TS lines(related to FIG. 3e, f ). (FIG. 13M) Quantification of the speed whenmobile of Dlxi1/2b::eGFP cells in fused hSS-hCS in TS and Ctrl (Ctrl:n=21 cells from 3 hiPSC lines from 3 subjects; TS: n=29 cells from 3hiPSC lines from 3 subjects; TS-Ctrl hybrid: n=12 from 3 hiPSC linecombinations; one-way ANOVA with Dunnett's multiple comparison test;***P<0.001). (FIG. 13N) Electroporation of the TS- and WT-CaV1.2channels into slices of mouse E14 ganglionic eminences (GE). (FIG. 13O)Representative example of time-lapse live imaging depicting thesaltatory migration of GFP+ cells in slices electroporated with CAG::GFPand either the WT- or the TS-CACNA1C. (FIG. 13P, 13Q) Quantification ofthe number of saltations (t-test; **P<0.01) and saltation length(t-test; ***P<0.0001) of GFP+ cells in electroporated mouse forebrainslices (n=33 cells for WT, n=23 cells for TS; from 3 litters). (FIG.13R) Scheme illustrating pharmacological manipulation of LTCC duringlive imaging of fused hSS-hCS in TS versus Ctrl. (FIG. 13S)Quantification of speed when mobile following exposure to the LTCCantagonist nimodipine (5 μM) in TS versus control (paired t-tests; Ctrl:n=13 cells from 3 hiPSC lines from 3 subjects, ***P<0.001; TS: n=12cells from 2 hiPSC lines from 2 subjects, **P<0.005). (FIG. 13T)Quantification of saltation length following exposure to roscovitine (15μM) in TS versus Ctrl (paired t-tests; Ctrl: n=7 cells from 2 hiPSClines from 2 subject, **P<0.005; TS: n=12 cells from 2 hiPSC lines from2 subjects; ***P<0.001). (FIG. 13U) Quantification of speed when mobilefollowing exposure to roscovitine (15 μM) in TS versus control (pairedt-tests; Ctrl: n=9 cells from 2 hiPSC lines from 2 subject, ***P<0.001;TS: n=12 cells from 2 hiPSC lines from 2 subjects; P=0.05).

FIG. 14A-14F. Characterization of Dlxi1/2b::eGFP+ cells after migration(FIG. 14A) Representative images of 3D-reconstructed Dlxi1/2b::eGFP+cell morphologies before and after migration from hSS into hCS. (FIG.14B) Representative examples of action potentials (slice recordings) inDlxi1/2b::eGFP+ cells in unfused hSS, in hSS of fused hSS-hCS and in hCSafter migration in fused hSS-hCS. (FIG. 14C) Array tomography (AT)showing expression of the GABAergic synapse marker GPHN colocalized withSYN1 in hCS of fused hSS-hCS but not in unfused hCS, while theglutamatergic marker PSD95 colocalized with SYN1 is found in both fusedand unfused hCS (equal volumes 1.2 μm deep). (FIG. 14D) Representativeexamples of whole-cell voltage clamp recordings of IPSCs and EPSCs fromDlxi1/2b::eGFP+ cells in unf used hSS, in fused hCS-hSS, or aftermigration in hCS (FIG. 14E) Representative examples of whole-cellvoltage clamp recordings of IPSCs and EPSCs in cells recorded fromunfused hCS cells and fused hCS cells. (FIG. 14F) Average peri-stimulussynaptic events (IPSCs and EPSCs) in Dlxi1/2::eGFP+ cells recorded inthe hCS side of fused hSS-hCS before and after electrical stimulation(paired t-test, *P<0.05).

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particular compositionsand methods described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “areprogramming factor polypeptide” includes a plurality of suchpolypeptides, and reference to “the induced pluripotent stem cells”includes reference to one or more induced pluripotent stem cells andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

By “pluripotency” and pluripotent stem cells it is meant that such cellshave the ability to differentiate into all types of cells in anorganism. The term “induced pluripotent stem cell” encompassespluripotent cells, that, like embryonic stem cells (hESC), can becultured over a long period of time while maintaining the ability todifferentiate into all types of cells in an organism. hiPSC have a humanhESC-like morphology, growing as flat colonies containing cells withlarge nucleo-cytoplasmic ratios, defined borders and prominent nuclei.In addition, hiPSC express pluripotency markers known by one of ordinaryskill in the art, including but not limited to alkaline phosphatase,SSEA3, SSEA4, SOX2, OCT3/4, NANOG, TRA-1-60, TRA-1-81, etc. In addition,the hiPSC are capable of forming teratomas and are capable of forming orcontributing to ectoderm, mesoderm, or endoderm tissues in a livingorganism.

As used herein, “reprogramming factors” refers to one or more, i.e. acocktail, of biologically active factors that act on a cell, therebyreprogramming a cell to multipotency or to pluripotency. Reprogrammingfactors may be provided to the cells, e.g. cells from an individual witha family history or genetic make-up of interest for heart disease suchas fibroblasts, adipocytes, etc.; individually or as a singlecomposition, that is, as a premixed composition, of reprogrammingfactors. The factors may be provided at the same molar ratio or atdifferent molar ratios. The factors may be provided once or multipletimes in the course of culturing the cells of the subject invention. Insome embodiments the reprogramming factor is a transcription factor,including without limitation, OCT3/4; SOX2; KLF4; c-MYC; NANOG; andLIN-28.

Somatic cells are contacted with reprogramming factors, as definedabove, in a combination and quantity sufficient to reprogram the cell topluripotency. Reprogramming factors may be provided to the somatic cellsindividually or as a single composition, that is, as a premixedcomposition, of reprogramming factors. In some embodiments thereprogramming factors are provided as a plurality of coding sequences ona vector. The somatic cells may be fibroblasts, adipocytes, stromalcells, and the like, as known in the art. Somatic cells or hiPSC can beobtained from cell banks, from normal donors, from individuals having aneurologic or psychiatric disease of interest, etc.

Following induction of pluripotency, hiPSC are cultured according to anyconvenient method, e.g. on irradiated feeder cells and commerciallyavailable medium. The hiPSC can be dissociated from feeders by digestingwith protease, e.g. dispase, preferably at a concentration and for aperiod of time sufficient to detach intact colonies of pluripotent stemcells from the layer of feeders. The spheroids can also be generatedfrom hiPSC grown in feeder-free conditions, by dissociation into asingle cell suspension and aggregation using various approaches,including centrifugation in plates, etc.

Genes may be introduced into the somatic cells or the hiPSC derivedtherefrom for a variety of purposes, e.g. to replace genes having a lossof function mutation, provide marker genes, etc. Alternatively, vectorsare introduced that express antisense mRNA, siRNA, ribozymes, etc.thereby blocking expression of an undesired gene. Other methods of genetherapy are the introduction of drug resistance genes to enable normalprogenitor cells to have an advantage and be subject to selectivepressure, for example the multiple drug resistance gene (MDR), oranti-apoptosis genes, such as BCL-2. Various techniques known in the artmay be used to introduce nucleic acids into the target cells, e.g.electroporation, calcium precipitated DNA, fusion, transfection,lipofection, infection and the like, as discussed above. The particularmanner in which the DNA is introduced is not critical to the practice ofthe invention.

Disease-associated or disease-causing genotypes can be generated inhealthy hiPSC through targeted genetic manipulation (CRISPR/CAS9, etc)or hIPSC can be derived from individual patients that carry adisease-related genotype or are diagnosed with a disease, e.g. TimothySyndrome cells exemplified herein. Moreover, neural diseases with lessdefined or without genetic components can be studied within the modelsystem. Conditions of neurodevelopmental and neuropsychiatric disordersand neural diseases that have strong genetic components or are directlycaused by genetic or genomic alterations can be modeled with the systemsof the invention. Genetic alterations include for example pointmutations in genes such as NLGN1/3/4, NRXN1/4, SHANK1/2/3, GRIN2B/A,FMR1, or CHD8 that represent risk alleles for autism spectrum disorders,point mutations in or deletions of genes such as CACNA1C, CACNB2,NLGN4X, LAMA2, DPYD, TRRAP, MMP16, NRXN1 or NIPAL3 that are associatedwith schizophrenia or autism spectrum disorders (ASD), etc, a tripletexpansion in the HTT gene that cause to Huntington's disease (HD),monoallelic mutations in genes such as SNCA, LRRK2 and biallelicmutations in genes such as PINK1, DJ-1, or ATP13A2 that predispose toParkinson disease (PD), single nucleotide polymorphisms (SNPs) in genessuch as ApoE, APP, and PSEN1/2 that confer risks for developingAlzheimer's disease (AD) and other forms of dementia, as well as SNPs ingenes such as CACNA1C, CACNB3, ODZ4, ANK3 that are associated withbipolar disease (BP); Angelman (UBE3A), Rett (MEPC2), Tuberous sclerosis(TSC1/2). Genomic alterations include copy number variations (CNVs) suchas deletions or duplications of 1q21.1, 7q11.23, 15q11.2, 15q13.3,22q11.2 or 16p11.2, 16p13.3 that are associated with ASD, schizophrenia,intellectual disability, epilepsy, etc; trisomy 21 and Down Syndrome,Fragile X syndrome caused by alteration of the FMR1 gene. Any number ofneurodevelopment disorders with a defined genetic etiology can beadditionally modeled by introducing mutations in or completely removingdisease-relevant gene(s) in control hiPSC using genome editing, e.g.CRISPR. A particular advantage of this method is that fact that editedhiPSC lines share the same genetic background as their corresponding,non-edited hiPSC lines. This reduces variability associated withline-line differences in genetic background.

Disease relevance. The effect of drugs on neurons or glial cells (e.g,astrocytes) is of particular interest, where efficacy and toxicity mayrest in sophisticated analysis of neuronal migratory and electricalinteractions, or the ability of neurons to form functional networks,rather than on simple viability assays. The discrepancy between thenumber of lead compounds in clinical development and approved drugs maypartially be a result of the methods used to generate the leads andhighlights the need for new technology to obtain more detailed andphysiologically relevant information on cellular processes in normal anddiseased states.

A number of important clinical conditions are associated with alteredneuronal or glial function, including neurodegenerative disorders(Alzheimer's disease (AD), Parkinson's disease (PD), Huntington'sdisease (HD), and amyotrophic lateral sclerosis (ALS), or psychiatricconditions such as schizophrenia and other psychoses, bipolar disorders,mood disorders, intellectual disability or autism spectrum disorders.

Genetic changes may include genotypes that affect migratory, or synapticfunction in disorders such as schizophrenia, autism spectrum disorders,monogenic disorders such as Timothy Syndrome, etc. In other embodimentseffects of genotypes and agents on neural development can be assessedusing neural cultures of the invention during the process ofdifferentiation and migration. Thus, neurodevelopmental effects can betested that either immediately affect function, maturation, andviability of developing cells, or exhibit long-term effects emerging asphenotypes in mature neural cultures.

A number of neuropsychiatric disorders may arise from an alteration ordisruption in the balance of excitation and inhibition in the cerebralcortical circuitry. Additionally, a number of studies have shown thatthe lack of proper cortical interneuron specification may play asignificant role in the development of neuropsychiatric disorders(schizophrenia, autism spectrum disorders, epilepsy and other seizuredisorders). This may entail a deviation from either the course ofinterneuron development, or aberrant transcriptional regulation in thecortical interneuron specification process. Understanding the effects ofboth GABAergic neurotransmission, alterations in inhibitory corticalcircuits, and how they may be responsible for the clinical featuresobserved in schizophrenia or autism are paramount to this field ofresearch.

Conditions of interest may also include DISC1-related disorders, Rettsyndrome, Fragile X, Alexander's disease, and others.

Autism spectrum disorders (ASDs) are neurodevelopmental disorderscharacterized by varying degrees of impaired social interaction andcommunication and the presence of repetitive and stereotypicalbehaviors. Some models of ASD emphasize the idea that abnormal synapsedevelopment underlies many features of the disease and postulateabnormalities in excitatory-inhibitory balance (E/I ratio). A betterunderstanding of neuronal interactions in ASDs will shed light onpathogenesis and the development of new treatment strategies.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

Neuronal migration is one of the fundamental mechanisms underlying thewiring of the brain. The nervous system grows both in size andcomplexity by using migration as a strategy to position cell types fromdifferent origins into specific coordinates, allowing for the generationof neural circuits. The migration of newly born neurons is a preciselyregulated process that is critical for the development of brainarchitecture. Neurons arise from the proliferative epithelium thatcovers the ventricular space throughout the neural tube (VZ, SVZ, oSVZ).During radial migration, neurons follow a trajectory that isperpendicular to the ventricular surface, moving alongside radial glialfibers expanding the thickness of the neural tube. In contrast,tangentially migrating neurons, often born in other ventricular regionsof the CNS (e.g, subpallium/ventral forebrain) move in trajectories thatare parallel to the ventricular surface and orthogonal radial glia.

The adult cerebral cortex contains two main classes of neurons:glutamatergic cortical neurons (also known as pyramidal cells) andGABAergic interneurons. Pyramidal cells are generated in the pallium—theroof of the telencephalon (dorsal forebrain)—and reach their finalposition by radial migration. In contrast, cortical interneurons areborn in the subpallium—the base of telencephalon (ventral forebrain)—andreach the cerebral cortex through a long tangential migration.

The layers of the cerebral cortex are generated in an “inside-out”sequence, with deep layers being generated first and superficial layerneurons being generated last. In parallel to this process, GABAergicinterneurons migrate to the cortical plate, where they dispersetangentially via highly stereotyped routes in the MZ, SP, and lowerintermediate zone/subventricular zone (IZ/SVZ). Interneurons then switchfrom tangential to radial migration to adopt their final laminarposition in the cerebral cortex.

The movement of cortical interneurons is saltatory. First, the cellextends a leading process. Second, the nucleus translocates towards theleading process, a step referred to as nucleokinesis and leads to thenet movement of the cell.

The translocation of the nucleus into the leading process is themechanism that best defines this type of saltatory neuronal migration.First, a cytoplasmic swelling forms in the leading process, immediatelyproximal to the nucleus. The centrosome, which is normally positioned infront of the nucleus, moves into this swelling. The centrosome isaccompanied by additional organelles, including the Golgi apparatus,mitochondria, and the rough endoplasmic reticulum. Second, the nucleusfollows the centrosome. These two steps are repeated producing thetypical saltatory movement of migrating neurons.

Tangentially migrating neurons do not always follow radial glial fibers.In general, tangentially migrating cells can move in clusters orindividually. Cellular interactions also differ depending on the natureof the substrate. They can be homotypic, when interactions occur betweencells of the same class, or heterotypic, when migrating cells rely onthe contact with other cell types for their migration or theirsubstrates. In the most common scenario, neurons respond to cues presentin the extracellular matrix or in the surface of other cells to achievedirectional migration.

GABAergic interneurons are inhibitory neurons of the nervous system thatplay a vital role in neural circuitry and activity. They are so nameddue to their release of the neurotransmitter gamma-aminobutyric acid(GABA). An interneuron is a specialized type of neuron whose primaryrole is to modulate the activity of other neurons in a neural network.Cortical interneurons are so named for their localization in thecerebral cortex.

There are interneuron subtypes categorized based on the surface markersthey express, including parvalbumin (PV)-expressing interneurons,somatostatin (SST)-expressing interneurons, VIP-expressing, serotoninreceptor 5HT3a (5HT3aR)-expressing interneurons, etc. Although theseinterneurons are localized in their respective layers of the cerebralcortex, they are generated in various subpallial locations.

Morphologically speaking, cortical interneurons may be described withregard to their soma, dendrites, axons, and the synaptic connectionsthey make. Molecular features include transcription factors,neuropeptides, calcium-binding proteins, and receptors theseinterneurons express, among many others. Physiological characteristicsinclude firing pattern, action potential measurements, passive orsubthreshold parameters, and postsynaptic responses, to name a few.

The PV interneuron group represents approximately 40% of the GABAergiccortical interneuron population. This population of interneuronspossesses a fast-spiking pattern, and fire sustained high-frequencytrains of brief action potentials. Additionally, these interneuronspossess the lowest input resistance and the fastest membrane timeconstant of all interneurons. Two types of PV-interneurons make up thePV interneuron group: basket cells, which make synapses at the soma andproximal dendrite of target neurons, and usually have multipolarmorphology and chandelier cells, which target the axon initial segmentof pyramidal neurons.

The SST-expressing interneuron group is the second-largest interneurongroup. SST-positive interneurons are known as Martinotti cells, andpossess ascending axons that arborize layer I and establish synapsesonto the dendritic tufts of pyramidal neurons. Martinotti cells arefound throughout cortical layers II-VI, but are most abundant in layerV. These interneurons function by exhibiting a regular adapting firingpattern but also may initially fire bursts of two or more spikes on slowdepolarizing humps when depolarized from hyperpolarized potentials. Incontrast to PV-positive interneurons, excitatory inputs onto Martinotticells are strongly facilitating.

The third group of GABAergic cortical interneurons is designated as the5HT3aR interneuron group. VIP-expressing interneurons are localized incortical layers II and III. VIP interneurons generally make synapsesonto dendrites, and some have been observed to target otherinterneurons. Relative to all cortical interneurons, VIP interneuronspossess a very high input resistance. In general they possess a bipolar,bitufted and multipolar morphology. Irregular spiking interneuronspossess a vertically oriented, descending axon that extends to deepercortical layers, and have an irregular firing pattern that ischaracterized by action potentials occurring irregularly duringdepolarizations near threshold, and express the calcium-binding proteincalretinin (CR). Other subtypes include rapid-adapting, fast-adaptingneurons IS2, as well as a minor population of VIP-positive basket cellswith regular, bursting, or irregular-spiking firing patterns. Of theVIP-negative 5HT3aR group, nearly 80% express the interneuron markerReelin. Neurogliaform cells are a type of cortical interneuron thatbelongs to this category: they are also known as spiderweb cells andexpress neuropeptide Y (NPY), with multiple dendrites radiating from around soma.

A transcriptional network plays a role in regulating proper developmentand specification of GABAergic cortical interneurons, including DLXhomeobox genes, LHX6, SOX6 and NKX2-1, LHX8, GSX1, GSX2. The DLX familyof homeobox genes, specifically DLX1, DLX2, DLX5, and DLX6, also play arole in the specification of interneuron progenitors, and are expressedin most subpallial neural progenitor cells.

Glutamatergic neurons. The mature cerebral cortex harbors aheterogeneous population of glutamatergic neurons, organized into ahighly intricate histological architecture. So-called excitatory neuronsare usually classified according to the lamina where their soma islocated, specific combinations of gene expression, by dendriticmorphologies, electrophysiological properties, etc.

Based on the differences in connections, pyramidal neurons areclassified as projection neurons with long axons that connect differentcortical regions or project to subcortical targets. Cortical projectionneurons can be further classified by hodology in associative,commissural and corticofugal subtypes. Associative projection neuronsextend axons within a single hemisphere, whereas commissural projectionneurons connect neurons in the two cortical hemispheres either throughthe corpus callosum or the anterior commissure. Cortifugal projectionneurons send axons to target areas outside the cerebral cortex, such asthe thalamus (corticothalamic neurons), pons (corticopontine neurons(CPN), spinal cord (costicospinal neurons), superior colliculus(corticotectal neurons) and striatum (corticostriatal neurons).

The terms “astrocytic cell,” “astrocyte,” etc. encompass cells of theastrocyte lineage, i.e. glial progenitor cells, astrocyte precursorcells, and mature astrocytes, which for the purposes of the presentinvention arise from a non-astrocytic cell by experimental manipulation.Astrocytes can be identified by markers specific for cells of theastrocyte lineage, e.g. GFAP, ALDH1L1, AQP4, EAAT1 and EAAT2, etc.Markers of reactive astrocytes include S100, VIM, LCN2, FGFR3 and thelike. Astrocytes may have characteristics of functional astrocytes, thatis, they may have the capacity of promoting synaptogenesis in primaryneuronal cultures; of accumulating glycogen granules in processes; ofphagocytosing synapses; and the like. A “astrocyte precursor” is definedas a cell that is capable of giving rise to progeny that includeastrocytes.

Astrocytes are the most numerous and diverse neuroglial cells in theCNS. An archetypal morphological feature of astrocytes is theirexpression of intermediate filaments, which form the cytoskeleton. Themain types of astroglial intermediate filament proteins are glialfibrillary acidic protein (GFAP) and vimentin; expression of GFAP,ALDH1L1 and/or AQP4P are commonly used as a specific marker for theidentification of astrocytes.

The functions of astroglial cells are many: astrocytes create the brainenvironment, build up the micro-architecture of the brain parenchyma,integrate neural circuitry with local blood flow and metabolic support,maintain brain homeostasis, store and distribute energy substrates,control the development of neural cells, synaptogenesis and synapticmaintenance and provide for brain defense. As such, there isconsiderable interest in studying the effects of drugs and othertherapeutic regimens on astrocytic cells.

Astroglia regulate formation, maturation, maintenance, and stability ofsynapses, thus controlling the connectivity of neuronal circuits.Astrocytes secrete numerous factors required for synaptogenesis.Synaptic formation depends on cholesterol produced and secreted byastrocytes. Glial cells also affect synaptogenesis through signalsinfluencing the expression of agrin and thrombin. Subsequently,astrocytes control maturation of synapses through several signalingsystems, which affect the postsynaptic density, for example bycontrolling the density of postsynaptic receptors. Astroglia factorsthat affect synapse maturation include TNF and activity-dependentneurotrophic factor (ADNF). Astrocytes may also limit the number ofsynapses.

Astrocytes and other glial cells can release a variety of transmittersinto the extracellular space, including glutamate, ATP, GABA andD-serine.

Astrocytes are involved in all types of brain pathologies from acutelesions (trauma or stroke) to chronic neurodegenerative processes (suchas Alexander's disease, Alzheimer's disease, Parkinson's disease,multiple sclerosis and many others) and psychiatric diseases(schizophrenia, autism spectrum disorders, etc). Pathologically relevantneuroglial processes include various programs of activation, which areessential for limiting the areas of damage, producing neuro-immuneresponses and for the post-insult remodeling and recovery of neuralfunction. Astroglial degeneration and atrophy in the early stages ofvarious neurodegenerative disorders may be important for cognitiveimpairments.

METHODS OF THE INVENTION

Methods are provided for the obtention and use of in vitro integratedforebrain systems, which comprise interacting cells of at least twoforebrain subdomains, including the dorsal pallium and ventralsubpallium. The methods comprise an initial step of differentiatingpluripotent cells, including without limitation induced humanpluripotent stem cells (hiPSC), into the forebrain subdomains of (i) aventral forebrain structure, referred to herein as a subpallial spheroid(hSS) comprising GABAergic interneurons; and (ii) a cerebral cortical,or dorsal pallium structure (hCS) comprising gluamatergic neurons. Thespheroids may also comprise neural progenitor cells, astrocytes, and thelike. Following this differentiation step, subpallial spheroid(s) (hSS)and cortical spheroid(s) (hCS) are placed adjacent to teach other inculture under conditions permissive for fusion or assembly of the twospheroids and generation of the integrated brain system with newproperties. In this case, the fused forebrain comprises functionallyintegrated neurons of excitatory and inhibitory types, which provides aplatform for analysis of the effect of agents on brain structure andfunction.

In some embodiments the neural cells are differentiated from inducedhuman pluripotent stem cells (hiPSC). In some embodiments the hiPSC arederived from somatic cells obtained from neurologically normalindividuals. In other embodiments the hiPSC are derived from somaticcells obtained from an individual comprising at least one alleleencoding a mutation associated with a neural disease.

Methods are also provided for determining the activity of a candidateagent on a disease-relevant integrated forebrain structure, the methodcomprising contacting the candidate agent with one or a panel of cellsor cell systems differentiated from human pluripotent stem cells, e.g.differentiated from hESC or from hiPSC, where the pluripotent stem cellsoptionally comprise at least one allele encoding a mutation associatedwith a neural disease; and determining the effect of the agent onmorphologic, genetic or functional parameters, including withoutlimitation formation of synapses, interneuron migration, and the like.

Generation of the subdomain spheroids and cells comprised thereinutilizes a multi-step process. Initially, hiPSC can be obtained from anyconvenient source, or can be generated from somatic cells usingart-recognized methods. The hiPSC are dissociated from feeders (or ifgrown in feeder free, aggregated in spheroids of specific sizes) andgrown in suspension culture in the absence of FGF2, preferably whendissociated as intact colonies. In certain embodiments the culture arefeeder layer free, e.g. when grown on vitronectin coated vessels. Theculture may further be free on non-human protein components, i.e.xeno-free, where the term has its usual art-recognized definition, forexample referring to culture medium that is free of non-human serum.Suspension growth optionally includes in the culture medium an effectivedose of a selective Rho-associated kinase (ROCK) inhibitor for theinitial period of culture, for up to about 6 hours, about 12 hours,about 18 hours, about 24 hours, about 36 hours, about 48 hours, (see,for example, Watanabe et al. (2007) Nature Biotechnology 25:681 686).Inhibitors useful for such purpose include, without limitation, Y-27632;Thiazovivin (Cell Res, 2013, 23(10):1187-200; Fasudil (HA-1077) HCl (JClin Invest, 2014, 124(9):3757-66); GSK429286A (Proc Natl Acad Sci USA,2014, 111(12):E1140-8); RKI-1447; AT13148; etc.

The suspension culture of hiPSC is then induced to a neural fate. Thisculture may be feeder-free and xeno-free. For hCS neural induction, aneffective dose of an inhibitor of BMP, and of TGFβ pathways is added tothe medium, for a period at least about 2 days, at least about 3 days,at least about 4 days, at least about 5 days, and up to about 10 days,up to about 9 days, up to about 8 days, up to about 7 days, up to about6 days, up to about 5 days. For example, dorsomorphin (DM) can be addedat an effective dose of at least about 0.1 μM, at least about 1 μM, atleast about 5 μM, at least about 10 μM, at least about 50 μM, up toabout 100 μM concentration, which inhibits bone morphogenetic protein(BMP) type I receptors (ALK2, ALK3 and ALK6). Other useful BMPinhibitors include, without limitation, A 83-01; DMH-1; K 02288; ML 347;SB 505124; etc. SB-431542 can be added at an effective dose of at leastabout 0.1 μM, at least about 1 μM, at least about 5 μM, at least about10 μM, at least about 50 μM, up to about 100 μM concentration, whichinhibits TGFβ signaling but has no effect on BMP signaling. Other usefulinhibitors of TGFβ include, without limitation, LDN-193189 (J ClinInvest, 2015, 125(2):796-808); Galunisertib (LY2157299) (Cancer Res,2014, 74(21):5963-77); LY2109761 (Toxicology, 2014, 326C:9-17); SB525334(Cell Signal, 2014, 26(12):3027-35); SD-208; EW-7197; Kartogenin; DMH1;LDN-212854; ML347; LDN-193189 HCl (Proc Natl Acad Sci USA, 2013,110(52):E5039-48); SB505124; Pirfenidone (Histochem Cell Biol, 2014,10.1007/s00418-014-1223-0); RepSox; K02288; Hesperetin; GW788388;LY364947, etc.

Generation of human sub-pallial spheroids (hSS) and cells comprisedtherein, including, for example neural progenitors, GABAergicinterneurons, astocytes etc. from somatic cells utilizes a similarmulti-step process with the inclusion of additional agents to promoteventral forebrain differentiation. Early spheroids patterned by SMADinhibition, e.g. at the time of transfer to the SMAD inhibitory medium,after about 12 hours, after about 24 hours, after about 1 day, afterabout 2 days, after about 3 days, after about 4 days, are cultured inthe presence of an effective dose of a Wnt inhibitor and an SHHinhibitor in the culture medium. The Wnt and SHH inhibitors aremaintained for a period of about 7 days, about 10 days, about 14 days,about 18 days, about 21 days, about 24 days, for example at aconcentration of from about 0.1 μM to about 100 μM, and may be fromabout 1 μM to about 50 μM, from about 5 μM to about 25 μM, etc.depending on the activity of the inhibitor that is selected.

Exemplary WNT inhibitors include, without limitation, XAV-939selectively inhibits Wnt/β-catenin-mediated transcription throughtankyrase1/2 inhibition with IC50 of 11 nM/4 nM in cell-free assays;ICG-001 antagonizes Wnt/β-catenin/TCF-mediated transcription andspecifically binds to element-binding protein (CBP) with IC50 of 3 μM;IWR-1-endo is a Wnt pathway inhibitor with IC50 of 180 nM in L-cellsexpressing Wnt3A, induces Axin2 protein levels and promotes β-cateninphosphorylation by stabilizing Axin-scaffolded destruction complexes;Wnt-C59 (C59) is a PORCN inhibitor for Wnt3A-mediated activation of amultimerized TCF-binding site driving luciferase with IC50 of 74 pM inHEK293 cells; LGK-974 is a potent and specific PORCN inhibitor, andinhibits Wnt signaling with IC50 of 0.4 nM in TM3 cells; KY02111promotes differentiation of hPSCs to cardiomyocytes by inhibiting Wntsignaling, may act downstream of APC and GSK3β; IWP-2 is an inhibitor ofWnt processing and secretion with IC50 of 27 nM in a cell-free assay,selective blockage of Porcn-mediated Wnt palmitoylation, does not affectWnt/β-catenin in general and displays no effect against Wnt-stimulatedcellular responses; IWP-L6 is a highly potent Porcn inhibitor with EC50of 0.5 nM; WIKI4 is a novel Tankyrase inhibitor with IC50 of 15 nM forTNKS2, and leads to inhibition of Wnt/beta-catenin signaling; FH535 is aWnt/β-catenin signaling inhibitor and also a dual PPARγ and PPARδantagonist.

SHH agonists include smoothened agonist, SAG, CAS 364590-63-6, whichmodulates the coupling of Smo with its downstream effector byinteracting with the Smo heptahelical domain (K_(D)=59 nM). SAG may beprovided in the medium at a concentration of from about 10 nM to about10 μM, from about 50 nM to about 1 μM, from about 75 nM to about 500 nM,and may be around about 100 nM.

Optionally the medium in this stage of the hSS culture process furthercomprises allopregnenolone from about day 10 to about day 23, e.g. fromday 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 until the conclusionof the stage; at a concentration of from about 10 nM to about 10 μM,from about 50 nM to about 1 μM, from about 75 nM to about 500 nM, andmay be around about 100 nM.

Optionally the hSS cultures are transiently exposed to retinoic acid,e.g. for about 1 to about 4 days, which may be from about day 10 toabout day 20, from about day 12 to about day 15, etc., at aconcentration of from about 10 nM to about 10 μM, from about 50 nM toabout 1 μM, from about 75 nM to about 500 nM, and may be around about100 nM.

For both hCS and hSS conditions, after about 5 days, about 6 days, about7 days, about 8 days, about 9 days, about 10, after about 15 days, afterabout 20 days, after about 25 days, e.g. around about 23 days, insuspension culture, the floating spheroids are moved to neural media todifferentiate neural progenitors. The media is supplemented with aneffective dose of FGF2 and EGF. The growth factors can be provided at aconcentration for each of at least about 0.5 ng/ml, at least about 1ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about20 ng/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100ng/ml.

To promote differentiation of neural progenitors into neurons, afterabout 1 week, about 2 weeks, about 3 weeks, about 4 weeks after FGF2/EGFexposure the neural medium is changed to replace the FGF2 and EGF withan effective dose of BDNF and NT3. The growth factors can be provided ata concentration for each of at least about 0.5 ng/ml, at least about 1ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about20 ng/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100ng/ml.

After about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks afterFGF2/EGF exposure, the spheres can be maintained for extended periods oftime in neural medium in the absence of growth factors, e.g. for periodsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. The number ofastrocytes in the cultures are initially low for the first month, andincrease in number after that, up to from about 5%, about 10%, about15%, about 20%, about 25%, to about 30% or more of the cells in thespheroids.

To fuse or assemble the two subdomain spheroids into an integratedforebrain structure, one or more of each hCS and hSS spheroids arebrought into close physical proximity, e.g. resting in a conical tube.The two spheroids may be combined in a 1:1 ratio, 1:2, 2:1, 1:3, 3:1,etc. ratio of hCS to hSS for the desired outcome. The integratedstructure is maintained in culture for extended periods of time, e.g.for up to about 30 days, up to about 40 days, up to about 50 days, up toabout 60 days, up to about 70 days, up to about 80 days, up to about 90days, or more. Other region-specific brain spheroids can be specifiedfrom hPSC in vitro and assembled using a similar approach to generatemulti-region brain 3D cultures that communicate and exhibit novel(emergent) features and capabilities versus conventional cultureapproaches or versus the same spheroids cultured in isolation.

Populations of cells can be isolated from the forebrain structure by anyconvenient method, including flow cytometry, magnetic immunoselection,immunopanning, etc. The cells thus isolated can be resuspended in anacceptable medium and maintained in culture, frozen, analyzed forparameters of interest; transplanted into a human or animal model; andthe like.

Screening Assays

In screening assays for the small molecules, the effect of adding acandidate agent to integrated forebrain system, to an hSS, to isolatedcells, and including without limitation at the initiation of fusionbetween the hCS and the hSS subdomains to determine the effect onmigration, synapse formation, etc. in culture is tested with one or apanel of cellular environments, where the cellular environment includesone or more of: electrical stimulation including alterations inionicity, stimulation with a candidate agent of interest, contact withother cells including without limitation neurons and neural progenitors,contact with infectious agents, e.g. Zika virus, and the like, and wherecells may vary in genotype, in prior exposure to an environment ofinterest, in the dose of agent that is provided, etc. Usually at leastone control is included, for example a negative control and a positivecontrol. Culture of cells is typically performed in a sterileenvironment, for example, at 37° C. in an incubator containing ahumidified 92-95% air/5-8% CO₂ atmosphere. Cell culture may be carriedout in nutrient mixtures containing undefined biological fluids such asfetal calf serum, or media which is fully defined and serum free. Theeffect of the altering of the environment is assessed by monitoringmultiple output parameters, including morphological, functional andgenetic changes.

Examples of analytic methods comprise, for example, assessing thesynaptic integration of migrated neurons by using array tomography todetect pre- and post-synaptic proteins in hCS before and after fusion tohSS, such as the presence of gephyrin (GPHN), a postsynaptic proteinlocalized to GABAergic synapses. To further examine these synapticpuncta ‘synaptograms’ consisting of a series of high-resolution sectionsthrough a single synapse may be obtained. Whole-cell voltage clamprecordings of synaptic responses can be performed on slices on theforebrain system, and to distinguish between excitatory postsynapticcurrents (EPSCs, downward deflecting) and IPSCs (upward deflecting), alow or solution may be used in the patch pipette with cells held at −40mV.

Live imaging of cells, including during cell migration, may be performedand cells modified to express a detectable marker. Calcium sensitivedyes can be used, e.g. Fura-2 calcium imaging; Fluo-4 calcium imaging,GCaMP6 calcium imaging, voltage imaging using voltage indicators such asvoltage-sensitive dyes (e.g. di-4-ANEPPS, di-8-ANEPPS, and RH237) and/orgenetically-encoded voltage indicators (e.g. ASAP1, Archer) can be usedon the intact spheroids, or on cells isolated therefrom.

Methods of analysis at the single cell level are also of interest, e.g.as described above: live imaging (including confocal or light-sheetmicroscopy), single cell gene expression or single cell RNA sequencing,calcium imaging, immunocytochemistry, patch-clamping, flow cytometry andthe like. Various parameters can be measured to determine the effect ofa drug or treatment on the forebrain system or cells derived therefrom.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can also be any cell component or cell product includingcell surface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Variability is expected and a range of values foreach of the set of test parameters will be obtained using standardstatistical methods with a common statistical method used to providesingle values.

Parameters of interest include detection of cytoplasmic, cell surface orsecreted biomolecules, frequently biopolymers, e.g. polypeptides,polysaccharides, polynucleotides, lipids, etc. Cell surface and secretedmolecules are a preferred parameter type as these mediate cellcommunication and cell effector responses and can be more readilyassayed. In one embodiment, parameters include specific epitopes.Epitopes are frequently identified using specific monoclonal antibodiesor receptor probes. In some cases the molecular entities comprising theepitope are from two or more substances and comprise a definedstructure; examples include combinatorically determined epitopesassociated with heterodimeric integrins. A parameter may be detection ofa specifically modified protein or oligosaccharide. A parameter may bedefined by a specific monoclonal antibody or a ligand or receptorbinding determinant.

Candidate agents of interest are biologically active agents thatencompass numerous chemical classes, primarily organic molecules, whichmay include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, select therapeutic antibodies and protein-basedtherapeutics, with preferred biological response functions. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins,Dermatology; and Toxicology, all incorporated herein by reference.

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term samples also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 to1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

As used herein, the term “genetic agent” refers to polynucleotides andanalogs thereof, which agents are tested in the screening assays of theinvention by addition of the genetic agent to a cell. The introductionof the genetic agent results in an alteration of the total geneticcomposition of the cell. Genetic agents such as DNA can result in anexperimentally introduced change in the genome of a cell, generallythrough the integration of the sequence into a chromosome, for exampleusing CRISPR mediated genomic engineering (see for example Shmakov etal. (2017) Nature Reviews Microbiology 15:169). Genetic changes can alsobe transient, where the exogenous sequence is not integrated but ismaintained as an episomal agents. Genetic agents, such as antisenseoligonucleotides, can also affect the expression of proteins withoutchanging the cell's genotype, by interfering with the transcription ortranslation of mRNA. The effect of a genetic agent is to increase ordecrease expression of one or more gene products in the cell.

Introduction of an expression vector encoding a polypeptide can be usedto express the encoded product in cells lacking the sequence, or toover-express the product. Various promoters can be used that areconstitutive or subject to external regulation, where in the lattersituation, one can turn on or off the transcription of a gene. Thesecoding sequences may include full-length cDNA or genomic clones,fragments derived therefrom, or chimeras that combine a naturallyoccurring sequence with functional or structural domains of other codingsequences. Alternatively, the introduced sequence may encode ananti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode adominant negative mutation, or dominant or constitutively activemutations of native sequences; altered regulatory sequences, etc.

Antisense and RNAi oligonucleotides can be chemically synthesized bymethods known in the art. Preferred oligonucleotides are chemicallymodified from the native phosphodiester structure, in order to increasetheir intracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases. Among usefulchanges in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity, e.g. morpholino oligonucleotide analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cells, in one or in a plurality ofenvironmental conditions, e.g. following stimulation with an agonist,following electric or mechanical stimulation, etc. The change inparameter readout in response to the agent is measured, desirablynormalized, and the resulting screening results may then be evaluated bycomparison to reference screening results, e.g. with cells having othermutations of interest, normal astrocytes, astrocytes derived from otherfamily members, and the like. The reference screening results mayinclude readouts in the presence and absence of different environmentalchanges, screening results obtained with other agents, which may or maynot include known drugs, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of selectedparameters, in addition to the functional parameters described above.For measuring the amount of a molecule that is present, a convenientmethod is to label a molecule with a detectable moiety, which may befluorescent, luminescent, radioactive, enzymatically active, etc.,particularly a molecule specific for binding to the parameter with highaffinity fluorescent moieties are readily available for labelingvirtually any biomolecule, structure, or cell type. Immunofluorescentmoieties can be directed to bind not only to specific proteins but alsospecific conformations, cleavage products, or site modifications likephosphorylation. Individual peptides and proteins can be engineered tofluoresce, e.g. by expressing them as green fluorescent protein chimerasinside cells (for a review see Jones et al. (1999) Trends Biotechnol.17(12):477-81). Thus, antibodies can be genetically modified to providea fluorescent dye as part of their structure

Depending upon the label chosen, parameters may be measured using otherthan fluorescent labels, using such immunoassay techniques asradioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.These techniques utilize specific antibodies as reporter molecules,which are particularly useful due to their high degree of specificityfor attaching to a single molecular target. U.S. Pat. No. 4,568,649describes ligand detection systems, which employ scintillation counting.These techniques are particularly useful for protein or modified proteinparameters or epitopes, or carbohydrate determinants. Cell readouts forproteins and other cell determinants can be obtained using fluorescentor otherwise tagged reporter molecules. Cell based ELISA or relatednon-enzymatic or fluorescence-based methods enable measurement of cellsurface parameters and secreted parameters. Capture ELISA and relatednon-enzymatic methods usually employ two specific antibodies or reportermolecules and are useful for measuring parameters in solution. Flowcytometry methods are useful for measuring cell surface andintracellular parameters, as well as shape change and granularity andfor analyses of beads used as antibody- or probe-linked reagents.Readouts from such assays may be the mean fluorescence associated withindividual fluorescent antibody-detected cell surface molecules orcytokines, or the average fluorescence intensity, the medianfluorescence intensity, the variance in fluorescence intensity, or somerelationship among these.

Both single cell multiparameter and multicell multiparameter multiplexassays, where input cell types are identified and parameters are read byquantitative imaging and fluorescence and confocal microscopy are usedin the art, see Confocal Microscopy Methods and Protocols (Methods inMolecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. Thesemethods are described in U.S. Pat. No. 5,989,833 issued Nov. 23, 1999.

Neuronal activity parameters. Of particular interest for the disclosedneuronal screening system are parameters related to the electricalproperties of the cells and therefore directly informative aboutneuronal function and activity. Methods to measure neuronal activity maysense the occurrence of action potentials (spikes). The characteristicsof the occurrence of a single spike or multiple spikes either in timelyclustered groups (bursts) or distributed over longer time (spike train)of a single neuron or a group of neurons indicate neuronal activationpatterns and thus reflect functional neuronal properties, which can bedescribed my multiple parameters. Such parameters can be used toquantify and describe changes in neuronal activity in the systems of theinvention.

Neuronal activity parameters include, without limitation, total numberof spikes (per recording period); mean firing rate (of spikes);inter-spike interval (distance between sequential spikes); total numberof bursts (per recording period); burst frequency; number of spikes perburst; burst duration (in milliseconds); inter-burst interval (distancebetween sequential bursts); burst percentage (the portion of spikesoccurring within a burst); total number of network bursts (spontaneoussynchronized network activity); network burst frequency; number ofspikes per network burst; network burst duration; inter-network-burstinterval; inter-spike interval within network bursts; network burstpercentage (the portion of bursts occurring within a network burst);salutatory migration, etc.

Quantitative readouts of neuronal activity parameters may includebaseline measurements in the absence of agents or a pre-defined geneticcontrol condition and test measurements in the presence of a single ormultiple agents or a genetic test condition. Furthermore, quantitativereadouts of neuronal activity parameters may include long-termrecordings and may therefore be used as a function of time (change ofparameter value). Readouts may be acquired either spontaneously or inresponse to or presence of stimulation or perturbation of the completeneuronal network or selected components of the network. The quantitativereadouts of neuronal activity parameters may further include a singledetermined value, the mean or median values of parallel, subsequent orreplicate measurements, the variance of the measurements, variousnormalizations, the cross-correlation between parallel measurements,etc. and every statistic used to a calculate a meaningful andinformative factor.

Comprehensive measurements of neuronal activity using electrical oroptical recordings of the parameters described herein may includespontaneous activity and activity in response to targeted electrical oroptical stimulation of all neuronal cells or a subpopulation of neuronalcells within the integrated forebrain. Furthermore, spontaneous orinduced neuronal activity can be measured in the self-assembledfunctional environment and circuitry of the neural culture or underconditions of selective perturbation or excitation of specificsubpopulations of neuronal cells as discussed above.

In the provided assays, comprehensive measurements of neuronal activitycan be conducted at different time points along neuronal maturation andusually include a baseline measurement directly before contacting theneural culture with the agents of interest and a subsequent measurementunder agent exposure. Moreover, long-term effects of agents on neuralmaturation and development can be assessed by contacting the immatureneural culture at an early time point with agents of interest andacquiring measurements of the same cultures after further maturation ata later time point compared to control cultures without prior agentexposure.

In some embodiments, standard recordings of neuronal activity of matureneural cultures are conducted after about 2 weeks, after about 3 weeks,after about 4 weeks, after about 6 weeks, after about 8 weeks followingfusion (i.e. after mixing the different subdomain components of theculture). Recordings of neuronal activity may encompass the measurementof additive, synergistic or opposing effects of agents that aresuccessively applied to the cultures, therefore the duration recordingperiods can be adjusted according to the specific requirements of theassay. In some embodiments the measurement of neuronal activity isperformed for a predetermined concentration of an agent of interest,whereas in other embodiments measurements of neuronal activity can beapplied for a range of concentrations of an agent of interest.

In some embodiments the provided assays are used to assess maturation ofthe neural culture or single components including GABAergicinterneurons, glutamatergic neurons, astrocytes, etc. Maturation ofneuronal cells can be measured based on morphology by opticallyassessing parameters such as dendritic arborization, axon elongation,total area of neuronal cell bodies, number of primary processes perneuron, total length of processes per neuron, number of branching pointsper primary process as well as density and size of synaptic punctastained by synaptic markers such as synapsin-1, synaptophysin, bassoon,PSD95, and Homer. Moreover, general neuronal maturation anddifferentiation can be assessed by measuring expression of markerproteins such as MAP2, TUJ-1, NeuN, Tau, PSA-NCAM, and SYN-1 alone or incombination using FACS analysis, immunoblotting, or fluorescencemicroscopy imaging, patch clamping. Maturation and differentiation ofneuronal subtypes can further be tested by measuring expression ofspecific proteins. For excitatory neuronal cells this includes stainingfor e.g. VGLUT1/2, GRIA1/2/3/4, GRIN1, GRIN2A/B, GPHN etc. Forinhibitory neuronal cells this includes staining for e.g. GABRA2,GABRB1, VGAT, and GAD67.

The results of an assay can be entered into a data processor to providea dataset. Algorithms are used for the comparison and analysis of dataobtained under different conditions. The effect of factors and agents isread out by determining changes in multiple parameters. The data willinclude the results from assay combinations with the agent(s), and mayalso include one or more of the control state, the simulated state, andthe results from other assay combinations using other agents orperformed under other conditions. For rapid and easy comparisons, theresults may be presented visually in a graph, and can include numbers,graphs, color representations, etc.

The dataset is prepared from values obtained by measuring parameters inthe presence and absence of different cells, e.g. genetically modifiedcells, cells cultured in the presence of specific factors or agents thataffect neuronal function, as well as comparing the presence of the agentof interest and at least one other state, usually the control state,which may include the state without agent or with a different agent. Theparameters include functional states such as synapse formation andcalcium ions in response to stimulation, whose levels vary in thepresence of the factors. Desirably, the results are normalized against astandard, usually a “control value or state,” to provide a normalizeddata set. Values obtained from test conditions can be normalized bysubtracting the unstimulated control values from the test values, anddividing the corrected test value by the corrected stimulated controlvalue. Other methods of normalization can also be used; and thelogarithm or other derivative of measured values or ratio of test tostimulated or other control values may be used. Data is normalized tocontrol data on the same cell type under control conditions, but adataset may comprise normalized data from one, two or multiple celltypes and assay conditions.

The dataset can comprise values of the levels of sets of parametersobtained under different assay combinations. Compilations are developedthat provide the values for a sufficient number of alternative assaycombinations to allow comparison of values.

A database can be compiled from sets of experiments, for example, adatabase can contain data obtained from a panel of assay combinations,with multiple different environmental changes, where each change can bea series of related compounds, or compounds representing differentclasses of molecules.

Mathematical systems can be used to compare datasets, and to providequantitative measures of similarities and differences between them. Forexample, the datasets can be analyzed by pattern recognition algorithmsor clustering methods (e.g. hierarchical or k-means clustering, etc.)that use statistical analysis (correlation coefficients, etc.) toquantify relatedness. These methods can be modified (by weighting,employing classification strategies, etc.) to optimize the ability of adataset to discriminate different functional effects. For example,individual parameters can be given more or less weight when analyzingthe dataset, in order to enhance the discriminatory ability of theanalysis. The effect of altering the weights assigned each parameter isassessed, and an iterative process is used to optimize pathway orcellular function discrimination.

The comparison of a dataset obtained from a test compound, and areference dataset(s) is accomplished by the use of suitable deductionprotocols, AI systems, statistical comparisons, etc. Preferably, thedataset is compared with a database of reference data. Similarity toreference data involving known pathway stimuli or inhibitors can providean initial indication of the cellular pathways targeted or altered bythe test stimulus or agent.

A reference database can be compiled. These databases may includereference data from panels that include known agents or combinations ofagents that target specific pathways, as well as references from theanalysis of cells treated under environmental conditions in which singleor multiple environmental conditions or parameters are removed orspecifically altered. Reference data may also be generated from panelscontaining cells with genetic constructs that selectively target ormodulate specific cellular pathways. In this way, a database isdeveloped that can reveal the contributions of individual pathways to acomplex response.

The effectiveness of pattern search algorithms in classification caninvolve the optimization of the number of parameters and assaycombinations. The disclosed techniques for selection of parametersprovide for computational requirements resulting in physiologicallyrelevant outputs. Moreover, these techniques for pre-filtering data sets(or potential data sets) using cell activity and disease-relevantbiological information improve the likelihood that the outputs returnedfrom database searches will be relevant to predicting agent mechanismsand in vivo agent effects.

For the development of an expert system for selection and classificationof biologically active drug compounds or other interventions, thefollowing procedures are employed. For every reference and test pattern,typically a data matrix is generated, where each point of the datamatrix corresponds to a readout from a parameter, where data for eachparameter may come from replicate determinations, e.g. multipleindividual cells of the same type. As previously described, a data pointmay be quantitative, semi-quantitative, or qualitative, depending on thenature of the parameter.

The readout may be a mean, average, median or the variance or otherstatistically or mathematically derived value associated with themeasurement. The parameter readout information may be further refined bydirect comparison with the corresponding reference readout. The absolutevalues obtained for each parameter under identical conditions willdisplay a variability that is inherent in live biological systems andalso reflects individual cellular variability as well as the variabilityinherent between individuals.

Classification rules are constructed from sets of training data (i.e.data matrices) obtained from multiple repeated experiments.Classification rules are selected as correctly identifying repeatedreference patterns and successfully distinguishing distinct referencepatterns. Classification rule-learning algorithms may include decisiontree methods, statistical methods, naive Bayesian algorithms, and thelike.

A knowledge database will be of sufficient complexity to permit noveltest data to be effectively identified and classified. Severalapproaches for generating a sufficiently encompassing set ofclassification patterns, and sufficiently powerfulmathematical/statistical methods for discriminating between them canaccomplish this.

The data from cells treated with specific drugs known to interact withparticular targets or pathways provide a more detailed set ofclassification readouts. Data generated from cells that are geneticallymodified using over-expression techniques and anti-sense techniques,permit testing the influence of individual genes on the phenotype.

A preferred knowledge database contains reference data from optimizedpanels of cells, environments and parameters. For complex environments,data reflecting small variations in the environment may also be includedin the knowledge database, e.g. environments where one or more factorsor cell types of interest are excluded or included or quantitativelyaltered in, for example, concentration or time of exposure, etc.

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, stem cell biology,human development and neurobiology. With respect to tissue culture andembryonic stem cells, the reader may wish to refer to Teratocarcinomasand embryonic stem cells: A practical approach (E. J. Robertson, ed.,IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M.Wasserman et al. eds., Academic Press 1993); Embryonic Stem CellDifferentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993);Properties and uses of Embryonic Stem Cells: Prospects for Applicationto Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil.Dev. 10:31, 1998).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXPERIMENTAL Example 1 Assembly of Functionally-Integrated ForebrainSpheroids from Human Pluripotent Cells to Study Development and Disease

The development of the central nervous system involves a coordinatedsuccession of events including the long-distance migration of GABAergicneurons from the ventral to the dorsal forebrain and their integrationinto cortical circuits. Defects in these processes have been associatedwith brain disorders and pluripotent stem cells (hPSC) hold promise indissecting the underlying pathophysiology in humans. However, theseinterregional interactions have not yet been modeled with human cells.Here, we describe an approach for generating from hPSCs neural 3Dspheroids resembling either the ventral forebrain and containingGABAergic interneurons—subpallial spheroids (hSS), or the dorsal palliumand containing glutamatergic neurons—cerebral cortical spheroids (hCS).We show that these subdomain-specific forebrain spheroids can beassembled in 3D in vitro to recapitulate the saltatory migration ofhuman interneurons into the cortex similar to migration in themid-gestation human fetal forebrain and to study interneuron dysfunctionin the context of human disease. Specifically, we found thatinterneurons derived from patients with Timothy syndrome—a severeneurodevelopmental disorder caused by a mutation in an L-type calciumchannel (LTCC), display more frequent but less efficient migratorysaltations, and that this deficit can be rescued pharmacologically invitro. Lastly, we demonstrate that after migration into hCS, humanGABAergic interneurons integrate synaptically with glutamatergic neuronsforming a 3D cortical microphysiological system (MPS) that exhibitsexcitatory and inhibitory synaptic activity. We anticipate that thisapproach will be useful for studying human development and themechanisms leading to neurodevelopmental disease, as well as forderiving spheroids resembling other human brain regions to ultimatelyassemble neural microcircuits in vitro.

The formation and function of the human cerebral cortex involves theassembly of circuits composed of glutamatergic excitatory neurons, whichare generated in the dorsal forebrain (pallium), and GABAergicinhibitory interneurons, which are born in the ventral forebrain(subpallium). After specification, interneurons migrate long distancesover several months during human fetal development and subsequentlyundergo activity-dependent maturation and integration into corticalcircuits. Genetic or environmental perturbations of this elaborateprocess can lead to an imbalance of cortical excitation and inhibitionand are thought to contribute to the pathophysiology of neuropsychiatricdisorders, including epilepsy and autism. These key developmentalprocesses, which occur mostly in mid to late gestation, have beenlargely inaccessible for functional studies in humans. Moreover, thedirected differentiation, and particularly the functional maturation ofhuman cortical interneurons from human pluripotent stem cells (inducedpluripotent stem cells, hiPSC, or embryonic stem cells, hESCs), hasbeen. To date, no reliable, personalized models exist to study themigration of human interneurons and their functional integration intocortical ensembles.

Here, we leverage a 3D differentiation approach using hPSCs to specifyneural spheroids resembling the pallium (hCS) or the subpallium (hSS),and we subsequently assemble them in vitro to model for the first timethe saltatory migration of human interneurons towards the cortex. We uselive imaging to show that this pattern of migration is similar tointerneuron migration in the mid-gestation human fetal forebrain. As aproof of principle, we examine patient-derived cells bearing a gain offunction mutation in CACNA1C, encoding the voltage gated L-type calciumchannel (LTCC) Cav1.2, and using a combination of live imaging andpharmacology in fused hSS-hCS, we find that interneuron migration isimpaired in spheroids from these patients. Lastly, we demonstrate thathiPSC-derived GABAergic interneurons migrating from hSS into hCS form acomplex functional network with cortical glutamatergic neurons, whichincludes both excitation and inhibition.

TABLE 1 SUPPLEMENTARY TABLE 1 INDIVIDUAL/ Ctrl hIPSC (5 subjects, 6lines) TS hIPSC (3 subjects, 7 lines) hESC EXPERIMENT 2242-1 8858-18858-3 1205-4 6593-8 H20961 8303-1 8303-2 7643-1 7643-5 7643-32 9862-29862-61 H9 hSS qPCR ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ characterization hSS ICC ✓✓ ✓ ✓ characterization hSS IHC ✓ ✓ ✓ ✓ ✓ ✓ characterization Single-cell✓ ✓ analysis Calcium ✓ ✓ ✓ ✓ ✓ ✓ ✓ Imaging - Fura-2 Calcium ✓ ✓ ✓ ✓Imaging - Fluo-4 DIxi1/2b::eGFP ✓ ✓ ✓ reporter characterization hSS-hCS✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ migration assays and pharmacol- ogy Dendriticbranch- ✓ ✓ ✓ ing Array Tomogra- ✓ ✓ phy Slice physiology ✓ ✓ ✓ ✓ ✓ ✓hSS culture on ✓ ✓ mouse forebrain slices

We have previously described the generation of floating, 3D neuralcultures from hPSCs resembling the dorsal pallium (hCS) that containboth deep and superficial layer cortical glutamatergic neurons, as wellas astrocytes. To specify spheroids resembling the ventral forebrain orthe subpallium (hSS), we exposed early spheroids patterned by doubleSMAD inhibition to small molecules modulating the WNT pathway (inhibitorof WNT production-2, IWP-2; 5 μM) and the SHH pathway (smoothenedagonist, SAG; 100 nM) in the presence of the growth factors FGF2 andEGF2 (20 μM) (hSS; FIG. 1a ; the 6 hiPSCs and 1 hESC line used in eachassay are shown in Table 1). At day 25 of hSS in vitro differentiation,we observed a strong induction of the transcription factor NKX2-1accompanied by high levels of FOXG1 expression and down-regulation ofthe dorsal pallial marker EMX1, suggestive of a ventral forebrain fate(FIG. 1b ). We next examined the cytoarchitecture in hSS cryosectionsand noticed that NKX2-1 was expressed in ventricular zone (VZ)-likestructures at day 25 (FIG. 1c ) but was distributed more broadly atlater stages (FIG. 1d ). At day 60, we observed strong expression byimmunocytochemistry of GABA and the GABA-synthesizing enzyme GAD67 inMAP2⁺ neurons (FIG. 1e, f ). Of the known markers of GABAergic subtypeidentity, somatostatin (SST), calretinin (CR) and calbindin (CB) werethe most strongly expressed, while at later stages (>200 days in vitro)and consistent with its later in vivo expression in development,parvalbumin (PV) was also present (FIG. 1g, h ; FIG. 5).

To more comprehensively characterize the population of cells in our 3Dcultures, we performed highly-parallel, genome-wide single celltranscriptional profiling at day 105 of in vitro differentiation usingnext-generation sequencing with stochastic barcoding18 (n=11,838 cellsfrom hCS and hSS; BD Resolve system; FIG. 1i ). Clustering andvisualization using the t-Distributed Stochastic Neighbor Embedding(t-SNE)19 projection of cells isolated from either hCS or hSS, showed aclear separation of the two conditions. Neurons expressing STMN2 werelocalized on the upper left side of the t-SNE space while progenitorsand mitotically active cells were distributed on the lower right side(FIG. 6a-c ). Close examination of the single cell data indicated thepresence of several subdomains in hCS (FIG. 1j, k ), including a groupof glutamatergic neurons (VGLUT1+) expressing the cortical layer markersTBR1, FEZF2, CTIP2; two groups of intermediate progenitors expressingTBR2, INSM1 and HES6; and a group of dorsal progenitors expressing LHX2,PAX6 and GLAST1 that also encompass HOPX+ outer radial glia-like cells(oRG). In contrast, hSS included a small group of oligodendrocyteprogenitors (OLIG2, SOX10) and a cluster of ventral neural progenitors,as well as a group of GABAergic postmitotic cells expressing DLX1, GAD1,SLC32A1, SCG2, SST STMN2 (Table 2 lists the genes in each cluster;patterns of expression for the top 25 genes in each cluster are shown inFIG. 6d-k ). Interestingly, astroglia from both hCS and hSS clusteredtogether and close to a small group of cells resembling the choroidplexus and expressing TTR and SLC13A4. Moreover, a very small group ofhCS-derived cells clustered with the GABAergic interneuron subdomain,and differential gene expression indicated that these cells expressedTBR1, RELN, PAX6 and CALB2. No cells of mesodermal or endodermal originwere found.

SUPPLEMENTARY TABLE 2 Gene Forward primer Reverse primer ASCL1TCTTCGCCCGAACTGATGC CAAAGCCCAGGTTGACCAACT BRACH TATGAGCCTCGAATCCACATAGTCCTCGTTCTGATAAGCAGTCAC CALB1 AGGGAATCAAAATGTGTGGGAAATCCTTCAGTAAAGCATCCAGTTC CALB2 TTTGCAGACAAGCCAGGATG TGTTTCACCGTGAACTGCACCHAT ACATGATTGAGCGCTGCATC ACTTGTCGTACCAGCGATTG CXCR4TGTTGGCTGAAAAGGTGGTC AACACAACCACCCACAAGTC CXCR7 ATCTTGAACCTGGCCATTGCTGTGTGACTTTGCACGTGAG DARPP32 ATCCTCACCCTGTTTTGTGC AGGTGGGCAAACAAGCAAACDLX1 ATGCACTGTTTACACTCGGC GACTGCACCGAACTGATGTAG DLX2ACGGGAAGCCAAAGAAAGTC TTTTGGAAACGCCGCTGAAG DLX5 TTCCAAGCTCCGTTCCAGACGAATCGGTAGCTGAAGACTCG EMX1 CGCAGGTGAAGGIGTGGTT TCCAGCUCTGCCGITTGT ERBB4AACAATGTGACGGCAGATGC TTCATGCAGGCAAAGCAGTC FOXG1 AACCTGTGTTGCGCAAATGCAAACACGGGCATATGACCAC GAD1 ATGCAACCAGATGTGTGCAG TGCCCTTTGCTTTCCACATCGAPDH CATGAGAAGTATGACAACAGCCT AGTCCTTCCACGATACCAAAGT GLI1ATGAAACTGACTGCCGTTGG ATGTGCTCGCTGTTGATGTG HOXB4 TCCTCGTTTTCAGCTTTGGCTCATTTGTTAGCGGGTGTCG LHX6 TGAGAGTCAGGTACAGTGCG GCCCATCCATATCGGCTTTGALMX1B AAACCCACGCAAACACACAC TCTCTTTCTGACAAGGCAGGAC MAFBTGGCCGGATGCATTTTTGAG AAGCACCATGCGGTTCATAC MEIS2 ACAGCTGGAGTGGCAAAAAGAAATTGTCAAGCCCCCGAAC NKX2-1 AGCACACGACTCCGTTCTC GCCCACTTTCTTGTAGCTTTCCNKX6-2 AGCACAAACCCTCGAACTTG CCCCCGGATTCTGCAAAAATAG NPYCGCTGCGACACTACATCAAC CAGGGTCTTCAAGCCGAGTT OCT3/4 CCCCAGGGCCCCATTTTGGTACCACCTCAGTTTGAATGCATGGGAGAGC OLIG2 GGACAAGCTAGGAGGCAGTGATGGCGATGTTGAGGTCGTG PVALB GGACAAAAGTGGCTTCATCGAG TCGTCAACCCCAATTTTGCCRAX1 GGCCATCCTGGGGTTTACC GGTCGAGGGGCTTCGTACT RELN TTITTGACGGCTTGCTGGTGTCCCAAATCCGAAAGCACTG SIX3 AGCAGAAGACGCATTGCTTC ACCAGTTGCCTACTTGTGTGSLC17A7 TGCGCAAGTTGATGAACTGC TCACGTTGAACCCAGAGATGG SOX17GTGGACCGCACGGAATTTG GGAGATTCACACCGGAGTCA SOX2 TTCACATGTCCCAGCACTACCAGATCACATGTGTGAGAGGGGCAGTGTGC

The sequences in the left column (forward primers) from top to bottomare set forth in SEQ ID NOs: 1-34. The sequences in the right column(reverse primers) from top to bottom are set forth in SEQ ID NOs: 35-68.

TABLE 3 Cluster Gene #1 Glutamatergic Neurons #1 BCL11B (CTIP2) #1BHLHE22 #1 CALB2 #1 CNTNAP2 #1 CRYM #1 FEZF2 #1 GAP43 #1 LMO3 #1 LMO7 #1LPL #1 MAP1B #1 MAPT #1 MEF2C #1 NEUROD2 #1 NEUROD6 #1 NFIB #1 NRN1 #1NSG2 #1 NTS #1 RTN1 #1 RUNX1T1 #1 SEZ6 #1 SLA #1 SLC17A7 (VGLUT1) #1SPHKAP #1 STMN2 #1 SYT1 #1 TBR1 #1 THSD7A #2 Intermediate Progenitors #2ASPM #2 CENPE #2 CENPF #2 EOMES (TBR2) #2 FAM64A #2 HES6 #2 HIST1H4C #2HMGB2 #2 INSM1 #2 MKI67 #2 NHLH1 #2 PRC1 #2 RRM2 #2 SMC4 #2 TOP2A #2TUBA1B #2 UBE2C #3 Radial Glia #3 AURKB #3 B3GAT2 #3 BIRC5 #3 C1orf61 #3CDK1 #3 CENPF #3 COL11A1 #3 DMRTA2 #3 EMX2 #3 FABP7 #3 FAM107A #3 FAM64A#3 FZD8 #3 GINS2 #3 HIST1H4C #3 HOPX #3 ID4 #3 KIAA0101 #3 KIF15 #3 LHX2#3 MCM4 #3 MKI67 #3 NUSAP1 #3 PAX6 #3 PRC1 #3 PTN #3 SFRP1 #3 SLC1A3(GLAST-1) #3 SMC4 #3 TOP2A #3 TPX2 #3 TUBA1B #3 VIM #4 Astroglia #4ANXA2 #4 AQP4 #4 ATP1A2 #4 B2M #4 BCAN #4 CLU #4 CRISPLD1 #4 CRYAB #4EDNRB #4 GPM6B #4 MLC1 #4 MT3 #4 NTN1 #4 NTRK2 #4 PI15 #4 PLTP #4 PMP2#4 PTGDS #4 RAB31 #4 RSPO3 #4 SPARC #4 SPARCL1 #4 SPON1 #4 TAGLN2 #4TIMP3 #4 TTYH1 #4 VCAM1 #4 VIM #5 Ventral Progenitors #5 BST2 #5 CRABP1#5 CRABP2 #5 DDIT4 #5 DPPA4 #5 ENO1 #5 FAM60A #5 FTL #5 HMGA2 #5 HMGB2#5 IGDCC3 #5 IRX2 #5 L1TD1 #5 LIN28A #5 LITAF #5 MDK #5 MIR302B #5 NMU#5 NR6A1 #5 PNP #5 PODXL #5 POU4F1 #5 PRDX6 #5 PRTG #5 QPRT #5 RPS6 #5TPM2 #5 TRIM71 #5 TUBA1C #6 GABAergic Neurons #6 BEX5 #6 C22orf42 #6CELF4 #6 CHGB #6 DIRAS3 #6 DLX1 #6 DLX5 #6 DLX6-AS1 #6 FGF14 #6 GAD1 #6GAP43 #6 ISL1 #6 LHX6 #6 LHX8 #6 MAPT #6 NEFL #6 NEFM #6 NEGR1 #6 NHLH2#6 NNAT #6 NPAS4 #6 NSG2 #6 ONECUT2 #6 ONECUT3 #6 PBX3 #6 PCDH17 #6 PCP4#6 PGM2L1 #6 POU2F2 #6 RELN #6 SCG2 #6 SCG5 #6 SIX3 #6 SLC32A1 #6 SPOCK2#6 SPOCK3 #6 SST #6 STMN2 #6 STMN4 #6 SYT4 #6 TENM2 #6 TSHZ2 #6 ZCCHC12#7 OPC #7 BCAN #7 BCAS1 #7 COL20A1 #7 EGFR #7 GPR17 #7 LHFPL3 #7 MBP #7NNAT #7 OLIG1 #7 OLIG2 #7 PDGFRA #7 PMP2 #7 RAB31 #7 S100B #7 SCRG1 #7SMOC1 #7 SOX10 #8 Choroid Plexus #8 CXCL14 #8 FOLR1 #8 IGF1 #8 IGFBP7 #8PCP4 #8 PMCH #8 RBM47 #8 RP11-395L14.4 #8 SLC13A4 #8 TRPM3 #8 TTR

We next explored the functional properties of hSS using calcium imaging(Fluo-4) at approximately day 50 of differentiation. We found that 7days of exposure to the neurosteroid and GABAA receptor agonistallopregnanolone (AlloP, 100 nM) combined with a short, 3-day exposureto retinoic acid (RA, 100 nM) (hSS-ISRA), significantly increased thefrequency of spontaneous calcium spikes (P=0.006; FIG. 7a-b ).Importantly, we found that exposure to AlloP with or without RA (hSS-ISAand hSS-ISRA, respectively) did not alter the subpallial fate, theneurotransmitter identity or the GABAergic subtypes in hSS (FIG. 8a-j ).As a result, these two conditions were primarily used for subsequentexperiments. In light of the observed spontaneous calcium activity andthe presence of astrocytes in our spheroids (FIG. 1j ), we investigatedsynaptogenesis in hSS using array tomography in 70 nm-thick sections. Wefound expression of the presynaptic protein synapsin-1 (SYN1) and thevesicular GABA transporter VGAT (FIG. 1l ). Lastly, we used whole-cellpatch clamping to record from neurons in 250 μm sections of hSS andfound that ˜75% of neurons generate action potentials in response todepolarization. At the same time, ˜60% of neurons exhibit spontaneousinhibitory postsynaptic responses (sIPSCs) that reverse in directionaround the chloride reversal potential and are abolished by the GABAAreceptor antagonist gabazine (10 μM) (FIG. 1m, n ; in contrast tosynaptic currents in hCS as shown in FIG. 9a ). Notably, the averageshape of sIPSCs recorded in hSS displayed a prolonged decay as comparedto the average EPSCs recorded from hCS, as is commonly observed incortical neurons20 (FIG. 9b ).

To develop a model for the migration of interneurons into the cerebralcortex, we placed one hCS and one hSS (˜day 60) adjacent to each otherinto a 1.5 ml conical microcentrifuge tube (FIG. 2a ). After 3 days thetwo spheroids fused (FIG. 2b ). For these experiments, we used hCS atday 60 of differentiation resembling dorsal pallium at mid-gestation16,a developmental stage characterized by extensive migration ofinterneurons in vivo. Moreover, we employed viral labeling of spheroidsbefore fusion to monitor cell migration; we used a previously describedultraconserved DNA element near the Dlx1 and Dlx2 locus (Dlxi1/2b) thatlabels the medial ganglionic eminences (MGE) and their derivatives21,22.Approximately 65% of Dlxi1/2b::eGFP cells in hSS expressed the GABAproducing enzyme GAD67 and they contained GABA and markers for GABAergicneuron subtypes by immunocytochemistry (FIG. 10a-d ). We then used liveimaging under environmentally controlled conditions (37° C., 5% CO2) tomonitor over several weeks the position of Dlxi1/2b::eGFP+ cells infused hSS-hCS. We observed a progressive movement of eGFP+ cells fromhSS into hCS over several weeks (FIG. 2c ). This movement was specificto fused hSS-hCS and unidirectional: we observed minimal movement bothfrom hCS into hSS in fused hSS-hCS and from hSS to hSS in fused hSS-hSS(FIG. 2d , FIG. 10e, f ). The same pattern of fusion and migration couldbe observed for hSS-hCS assembled between day 60-90 of differentiation(FIG. 10g ). Importantly, when hSS were plated on a coverslip themigration was inefficient or absent (FIG. 10h-j ), similar to what hasbeen reported in rodent cultures23. In the first 10 days after fusion,the vast majority of Dlxi1/2b::eGFP cells that migrated away from hSShad the leading process positioned towards hCS at either a 45° or 90°angle relative to the fusion interface (FIG. 10k ). After 30-50 dayspost-fusion, 60% of the migrated cells were localized within the outer100 μm of hCS (FIG. 10l ), and overall, a large population ofinterneurons migrated into hCS as shown by optical clearing with iDISCOand reconstruction (FIG. 2e ). Interestingly, we also observed processesof Dlxi1/2b::eGFP cells that briefly touched VZ-like regions containingprogenitors in hCS, reminiscent of the ventricle-directed migrationdescribed in rodents (FIG. 10m-o ). We next investigated the fate ofDlxi1/2b::eGFP cells in hSS and after 2 weeks of migration from hSS intohCS by isolating single cells from dissociated spheroids usingFluorescence Activated Cell Sorting (FACS) and Smart-seq2 fortranscriptome analysis (FIG. 11a ). We found that the majority ofmigrated cells expressed subpallial markers (DLX1, DLX2, DLX5, DLX6) andcortical interneuron markers (GAD1, GAD2, VGAT and CELF4) (FIG. 11b ).We found few cells expressing PAX6 or TH, which are indicative ofolfactory interneurons, or SP8, GSX2 or CHAT, which are indicative ofstriatal neurons, suggesting that the Dlxi1/2b reporter is primarilylabeling cortical interneurons in hSS (FIG. 11b ). Immunocytochemistryin fused hSS-hCS confirmed that the majority of migrated cells expressSST (FIG. 10p-s ).

We next used confocal imaging in 10-15 hrs long sessions to capture themovement of Dlxi1/2b::eGFP cells in fused hSS-hCS. Interneurons moved ina saltatory pattern followed by extensive pausing periods (FIG. 2f ).This characteristic, cyclical movement involved an extension of theleading process in one direction followed by a transient swelling of thesoma and nuclear translocation (FIG. 2g, h ). This pattern of migrationis similar to what has been observed in migrating interneurons inrodents25-27, although the ratio between the length of the leadingprocess and the diameter of the soma in human hSS-derived interneuronsis almost double the ratio in mouse interneurons, as quantified in mouseE18 slices imaged under similar conditions and using the same viralreporter (FIG. 12). To validate the pattern of interneuron migrationobserved in fused hSS-hCS, we performed live imaging of cells labeledwith the Dlxi1/2b::eGFP reporter in human mid-gestation forebrain tissue(gestational weeks, GW18 and GW20; FIG. 2i ). Dlxi1/2b::eGFP-labeledcells in fetal forebrain tissue co-expressed GABA and NKX2-1 (FIG. 12a-f) and we observed a similar cell morphology and cyclical pattern ofmigration that included long pausing periods and saltations followingnucleokinesis (FIG. 2j, k ; FIG. 12g-l ).

We tested whether we could pharmacologically manipulate interneuronmigration in fused hSS-hCS (FIG. 2l ). We imaged the movement ofDlxi1/2b::eGFP cells before and after exposure to a small moleculeantagonist of the CXCR4 receptor (AMD3100, 100 nM). This receptor isexpressed in hSS (FIG. 8j ) as well as in migrating interneurons, and ithas been shown to play a key role in the migration of corticalinterneurons by binding to a ligand secreted in the dorsal pallium.Blocking the CXCR4 receptor resulted in a significant reduction in thefrequency of saltations (FIG. 2m , P=0.03), the saltation length (FIG.2n , P=0.006), the speed when mobile (FIG. 2o , P=0.006), and a changein the path directness (FIG. 2p , P=0.02; FIG. 10t ).

We next investigated whether fused hSS-hCS could be used to modelmigration defects in neurodevelopmental disorders. Previous work inrodents has indicated that L-type voltage gated calcium channels (LTCC)play a critical role in interneuron migration by regulating thefrequency of saltations and, ultimately, migration termination23. LTCChave been repeatedly associated with neuropsychiatric disease ingenome-wide association studies31-33, and gain of function mutations inthe LTC-encoding CACNA1C gene lead to Timothy syndrome (TS)—a severeneurodevelopmental disorder characterized by autism spectrum disorderand epilepsy9,10. We generated hSS and hCS from hiPSC from 3 patientswith TS (7 hiPSC lines derived from 2 males and 1 female) carrying thesame p.G406R point mutation (FIG. 3a ) and compared them to 4 unaffectedcontrol subjects (4 hiPSC lines derived from 3 males and 1 female) (FIG.13a, b ; TS and control hiPSC lines used in various assays are shown inTable 1). We did not observe defects in the differentiation of TS hiPSClines into hSS as assessed by gene expression and immunocytochemistry(FIG. 13c-g ). Calcium imaging using the ratiometric dye Fura-2 showedincreased residual calcium following depolarization in hSS-derived TSneurons versus control neurons (FIG. 3b, c ; P<0.001), as well as inhCS-derived TS neurons compared to control cells (FIG. 13h ; P<0.001),similar to what we have previously shown in TS hiPSC-derivedglutamatergic neurons. We investigated the migration of Dlxi1/2b::eGFPcells in fused hSS-hCS (FIG. 3d ; FIG. 13i ) and found an increase inthe frequency of saltations in neurons from all three TS patients (FIG.3e ; P<0.001; data by hiPSC lines shown FIG. 13j ) in agreement with therole of calcium in promoting interneuron motility. Interestingly, thesaltation length (FIG. 3f ; P<0.001; data by hiPSC lines shown FIG. 13k) and the speed when mobile were reduced in TS as compared to controls(P<0.001; FIG. 13l ) resulting in an overall less efficient migration(FIG. 3g ; P<0.001). Moreover, this effect was cell-autonomous sincemigration of Dlxi1/2b::eGFP+ cells from TS-hSS into control-hCS did notinfluence the phenotype (FIG. 3e, f ; FIG. 13j-l ). To provideadditional support for these results, we electroporated cDNA encodingTS- and WT-CaV1.2 into slices of mouse E14 ganglionic eminences andperformed live imaging of GFP+ cells ˜48 hrs later

(FIG. 13m, n ). Consistent with our findings in TS hSS-hCS, we observeda defect in mouse TS-CaV1.2 electroporated neurons displaying morefrequent (FIG. 13o ; P<0.01) but shorter saltations (FIG. 13p ;P<0.001). To determine if the TS migratory phenotype was a result ofLTCC activity and could be reversed, we treated fused hSS-hCS with LTCCblockers during imaging (FIG. 3h, i ; FIG. 13q-t ). Previous work inrodents has shown that pharmacological manipulation of LTCC influencesinterneuron migration23, and we found that application of thedihydropyridine LTCC blocker nimodipine (5 μM) significantly reducedsaltation length and speed when mobile in control Dlxi1/2b::eGFP+ cells(P<0.001). However, the deficit in these parameters was rescued in TSDlxi1/2b::eGFP+ cells following exposure to the LTCC antagonist(P<0.001). Moreover, roscovitine, a cyclin-dependent kinase inhibitorthat increases voltage-dependent inactivation of CaV1.235,36 (15 μM),had a similar effect in rescuing saltation length in TS Dlxi1/2b::eGFP+cells (P<0.001). These results indicate that the migration defect ininterneurons carrying the TS gain-of-function mutation can be restoredby reducing the activity of LTCC, which likely results in a higherprobability and efficiency of Dlxi1/2b::eGFP saltations.

To investigate the hSS-derived neurons that migrated into the hCSnetwork, we examined their single cell transcriptome at 4 weeks afterhSS-hCS assembly using FACS and Smart-seq2 RNA-seq (n=181 single cells;FIG. 4a ). t-SNE analysis indicated 3 main clusters (A-C; FIG. 4b ),with Dlxi1/2b::eGFP+ cells in hSS distributed primarily in cluster A,while Dlxi1/2b::eGFP+ cells migrated into hCS primarily distributed inclusters B and C (FIG. 4 c; χ ²-test, χ²=43.39, P<0.0001). Cells in allclusters expressed similar levels of GAD1 and CELF4 (FIG. 11c ), butcluster B and C down-regulated the subpallial marker PBX3. Migratedcells displayed expression changes in genes previously associated withinterneuron migration such as ERBB4, NNAT, MALAT1, SOX11 and NXPH137,38(FIG. 4d ). Interestingly, migrated neurons also had higher levels ofseveral activity dependent genes, including FOS, the AMPA-receptortrafficking regulator GRIP239 and the growth factor IGF140, as well asgenes associated with neurodevelopmental disease (RASD141, TCF442) (FIG.4d ; FIG. 11c ; Table 4).

TABLE 4 A vs B and C B vs A and C C vs A and B Gene Name P value GeneName P value Gene Name P value MEIS2 0 SST 0 NNAT 0 NEFL 0 NNAT 0 UBB7.68E−14 HSPA8 1.11E−16 SOX11 0 RP11-138I1.4 2.97E−13 MAB21L2 3.55E−15YWHAQ 0 NDUFA4 8.78E−13 UCHL1 2.04E−14 CPE 0 TMSB10 1.27E−12 SOX4  3E−14 MEIS2 0 TINCR 1.33E−12 PGAM1 1.67E−13 TCF4 1.44E−15 LINC006891.25E−11 CDC42 1.69E−13 SOX4 3.66E−15 CALM2 1.57E−11 MAGED2 4.02E−13TMSB4XP8 5.33E−15 AC017104.2 2.65E−11 JAK3 4.49E−13 TMSB4X 5.88E−15RP5-1065J22.8 2.65E−11 CHN1 1.47E−12 VAT1L 1.21E−14 HSPA8 4.45E−11 PLD32.27E−12 ERBB4 1.17E−13 DLX6 4.78E−11 VDAC2 2.54E−12 SLC26A11 1.46E−13STMN1 5.32E−11 MDH1 2.68E−12 PLCXD1 2.16E−13 SAP30L-AS1 6.54E−11 SORL12.82E−12 SCG2 2.17E−13 ATG2A 1.12E−10 CPE  9.2E−12 RP11-55K13.1 3.03E−13SIX6 1.82E−10 COX5B 1.05E−11 NCALD 3.14E−13 SOX6 2.14E−10 RP11-82C23.21.36E−11 ATPIF1 4.23E−13 DOCK5 7.92E−10 LRCH4 1.43E−11 PLD3  2.5E−12RASD1 8.28E−10 TCF4 1.65E−11 AP000350.4 1.04E−11 GHR 9.24E−10 MUC201.67E−11 NEFL 1.84E−11 DLX6-AS2 1.35E−09 DLX6-AS1 1.94E−11 MTO1 2.04E−11ZFP64 1.97E−09 PTPRB 3.07E−11 HAT1 2.08E−11 TRABD2A 2.41E−09 PARK7 3.6E−11 ZNF486 2.38E−11 CHRNB1 2.49E−09 YWHAZ 6.62E−11 PGAM1 5.55E−11NFIA 2.63E−09 SST 9.22E−11 BCAS4 5.97E−11 FOS  2.8E−09 TTC39B  1.2E−10PCDH9 9.26E−11 LIPH 3.05E−09 DLX6  2.1E−10 CEP41 1.18E−10 RP11-814P5.13.32E−09 ATP5O 2.18E−10 LHX6 1.33E−10 MALAT1 3.72E−09 RDH13 2.23E−10GNG3 1.59E−10 RP11-379H18.1 5.15E−09 SLC25A53 2.58E−10 FAM189B  5.7E−10MPP3 5.25E−09 TMEM130 2.61E−10 ZNF114 6.01E−10 MAB21L2 5.97E−09 DLX6-AS2 2.7E−10 JUN 7.41E−10 NGRN 1.14E−08 CALM2 3.02E−10 BSCL2 8.47E−10CTC-250I14.6 0.000000018 SAP30L-AS1 4.29E−10 GREB1 1.21E−09 ZNF6552.12E−08 WDR31 5.26E−10 MUC20 1.41E−09 CTD-2293H3.1 2.46E−08 PLA2G4C5.57E−10 C16orf62 2.66E−09 NDC80 2.55E−08 PSMB5 6.99E−10 TMSB4XP43.15E−09 MRPS33 2.84E−08 GNG3 8.36E−10 MIF 3.31E−09 RNF152 2.87E−08GATA3 8.59E−10 HRK 3.79E−09 RBFOX2 3.43E−08 DLX5 8.75E−10 JDP2 3.98E−09SUOX 3.52E−08 RP11-128A17.1 8.95E−10 AC009403.2 4.13E−09 RORB 4.28E−08CDH6 1.08E−09 IGF1  5.7E−09 GFRA1 4.67E−08 PDE7B 1.24E−09 ATP1B16.48E−09 SCG2 5.47E−08 GTPBP2  1.4E−09 TCTA 6.93E−09 RGS16 5.54E−08 GOT11.46E−09 SORL1 8.69E−09 ROGDI 0.000000067 OPA3 1.61E−09 TTC39B 8.85E−09SOX4 0.000000103 CISD2 2.05E−09 EIF2AK2 9.74E−09 HRK 0.000000121 GATM2.07E−09 KB-1107E3.1 1.01E−08 NMS 0.000000125 NUDC 2.14E−09 PLS31.06E−08 EML6 0.00000015 NFIB 2.27E−09 TSPAN3 1.11E−08 YBX1 0.00000016CRABP1 2.34E−09 ACBD4 1.11E−08 SDCBP 0.000000161 RP11-977G19.11 2.82E−09PTPRG 1.11E−08 PPP1R17 0.000000212 ZNF114 2.87E−09 PKM 1.17E−08 ZNF2250.00000024 RP11-55K13.1 4.71E−09 MTHFR 1.21E−08 EP400NL 0.000000245MIEF2 4.97E−09 ENO2 1.32E−08 VPS29 0.000000277 PRRT3 5.39E−09 MYO15A1.43E−08 TOX 0.000000319 GHR 5.91E−09 UCHL1 1.45E−08 CYP20A1 0.000000364DTWD2 6.72E−09 STPG1 1.47E−08 FTH1P16 0.000000403 ANGPT2  7.1E−09 GRIP21.59E−08 BIRC5 0.000000414 PLIN2 7.18E−09 ZNF844 1.87E−08 ACTB0.000000458 ERBB4 7.32E−09 CHN1 1.89E−08 NETO2 0.000000521 PDE4C8.23E−09 STEAP4 3.19E−08 TIPIN 0.000000555 DLX1 8.38E−09 LINC003383.22E−08 CRTC3 0.000000587 AGT 9.16E−09 PDE7B 3.46E−08 EXOSC20.000000633 C19orf40 9.41E−09 TMEM130 3.49E−08 CABP7 0.000000693 RAB3B9.44E−09 CALCOCO2 4.28E−08 ATXN2 0.000000822 TCTA 1.01E−08 JAK3 4.74E−08TMEFF2 0.000000886 NACAD 1.04E−08 LGALS3BP 0.000000054 C8orf340.000000989 TMSB4X 1.19E−08 DENND2A 6.41E−08 NGRN 0.000000012 CDH236.48E−08 PCDH9 1.21E−08 SLC22A17 6.59E−08 HOTAIRM1 1.24E−08 MLTK6.97E−08 BSCL2 1.42E−08 GFRA1 7.13E−08 PPP1R13L 1.45E−08 TTL 7.31E−08LHX5 1.47E−08 CYCS 7.32E−08 TES 1.69E−08 AC104532.4 8.76E−08 CADM21.83E−08 FRMD4B 9.97E−08 TXNRD2 1.99E−08 TMEM42 0.0000001 STK17B2.29E−08 AC137932.1 0.000000103 DLX2 0.000000023 VDAC2 0.000000106 TMED32.43E−08 STMN4 0.000000108 SNAP25 2.49E−08 RP11-82C23.2 0.000000108BTBD10 2.75E−08 NXPH1 0.000000109 NEFM 2.78E−08 CADM1 0.000000136PPP2R3B 2.78E−08 POC1B 0.000000145 RWDD2A 2.89E−08 LDHBP1 0.000000145WDR19 2.93E−08 TACO1 0.000000151 LINC00338 3.21E−08 DUSP28 0.000000154ATG2A 3.42E−08 TSPYL2 0.000000155 MTA3 3.45E−08 CTC-359D24.3 0.000000162IFT20 3.55E−08 FKBP2 0.000000169 RWDD2B 3.91E−08 SLC16A3 0.000000176PSME2 0.000000042 PCDH9-AS1 0.000000178 AC040977.1 4.34E−08 APLP10.000000187 SOX11 4.36E−08 CALY 0.000000188 RAB7L1 4.42E−08 HYPK0.000000203 GAS6-AS2 4.88E−08 EHD4 0.000000215 ATP6V1D 5.47E−08 FOS0.000000218 ZNF486 5.53E−08 ERVMER34-1 0.000000222 APLP1 6.22E−08SLC25A34 0.000000258 CATSPER2 6.35E−08 ZNF154 0.000000295 CISD1 6.81E−08SFMBT2 0.00000031 AC004453.8 6.94E−08 RDH13 0.000000322 YWHAB 6.96E−08PARK7 0.00000035 ZNF772 7.78E−08 RBP1 0.00000035 RP5-1065J22.8 8.81E−08JUND 0.000000368 POR 9.15E−08 CLSPN 0.000000375 ALDOC 9.49E−08 SPARC0.000000376 ZC3H12B 0.000000104 LRCH4 0.000000401 ARF5 0.000000104 APAF10.000000413 SMTN 0.000000105 ZBTB3 0.000000421 NDUFC1 0.00000011 TWSG10.000000438 ATRN 0.000000116 LINC00689 0.000000441 BEX5 0.000000124 PAWR0.000000479 LINC00689 0.000000137 CAPZA1 0.000000513 ZNF385D 0.000000144TAPBP 0.000000529 GPX3 0.000000147 MAB21L1 0.000000563 LAMP5 0.000000151AHI1 0.000000565 JDP2 0.000000159 TMEM205 0.00000057 LINC011020.000000161 LIPH 0.000000591 SEPT4 0.000000162 ARHGAP44 0.000000598TACO1 0.000000163 SRP14-AS1 0.000000662 ATP5G1 0.000000167 CDC420.000000697 MICALL1 0.000000174 RP11-192H23.5 0.000000707 TUBA4A0.000000179 DYNC1I1 0.000000791 CLTB 0.000000181 PTPRF 0.000000816RP11-192H23.5 0.000000183 CLU 0.000000818 SEMA4C 0.000000187 PURB0.00000083 MEIS1 0.000000192 PRRT3 0.000000831 DYNLT1 0.000000199 PGRMC10.000000852 HSPA9 0.000000202 TES 0.00000089 TMSB4XP8 0.000000215 GOT10.000000899 CYCS 0.000000239 CX3CL1 0.000000981 TMEM120A 0.000000241ACTA2 0.000000268 SPOCK3 0.000000276 SUMF2 0.000000286 PBX3 0.000000288DOCK5 0.000000295 ZNF225 0.000000297 TMSB10 0.000000304 RNF1810.000000321 AC027763.2 0.000000338 C14orf166 0.000000358 FAIM20.00000037 CDH13 0.000000387 PSTPIP2 0.000000405 RP11-178G16.40.000000409 MGST3 0.00000041 IDH3B 0.000000414 GULP1 0.000000415 TMEM2160.000000417 PARVA 0.000000417 GS1-72M22.1 0.000000418 TMEM205 0.00000043NAP1L5 0.000000436 C21orf33 0.000000437 TMEM132B 0.000000447 WDR550.000000475 PSMD12 0.000000479 STMN4 0.000000483 CHRD 0.000000506 MOAP10.000000509 CTB-60E11.9 0.000000524 ATF7IP2 0.000000535 RNF5 0.000000536LRRC36 0.000000541 MRPL22 0.000000545 OTUD6B 0.000000557 TMEM106A0.000000566 PSMB1 0.000000581 SUMF1 0.000000585 PSMD13 0.000000611 RAB3C0.00000062 PGM2L1 0.000000633 DNAJC17 0.000000639 ZCCHC17 0.000000643PPA2 0.000000665 RBP1 0.000000668 PDP2 0.000000668 MLEC 0.000000694CARD14 0.000000696 ATP1B1 0.000000702 NDE1 0.000000709 TMED9 0.000000735KB-1107E3.1 0.000000743 CCDC167 0.000000758 ATP6V1A 0.00000077 B3GNT10.000000774 MATR3 0.000000808 TUBB2A 0.000000884 DNAH9 0.00000089 GPRC5A0.000000903 USP11 0.000000935 TUBB4A 0.000000984

To further investigate integration of migrated hSS-derived, we examinedthe dendritic morphology of Dlxi1/2b::eGFP+ cells in hSS and in fusedhSS-hCS. We found that the mostly bipolar hSS-derived cells that movedinto hCS increased the complexity of their dendritic branching (FIG. 4e,f ; P<0.001; FIG. 14a ). We then measured their electrical properties inhSS before and after fusion by quantifying action potential generationin response to steps of depolarizing current. We found thatDlxi1/2b::eGFP+ cells that had migrated into hCS had double the maximumaction potential generation rate as compared to Dlxi1/2b::eGFP+ cells inunf used hSS or to non-migrated Dlxi1/2b::eGFP+ cells in fused hSS-hCS(FIG. 4g ; P<0.001; FIG. 14b ). We then assessed the synapticintegration of migrated neurons by using array tomography to detect pre-and post-synaptic proteins in hCS before and after fusion to hSS, andobserved the presence of gephyrin (GPHN), a postsynaptic proteinlocalized to GABAergic synapses, in hCS fused to hSS but not unfused hCS(FIG. 14c ). To further examine these synaptic puncta in fused hSS-hCS,we constructed ‘synaptograms’ consisting of a series of high-resolutionsections through a single synapse, and found colocalization of eGFP fromDlxi1/2b labeled cells with the presynaptic proteins SYN1 and VGAT aswell as the postsynaptic protein GPHN (FIG. 4h-i ). To investigate thepresence of functional synapses in migrated Dlxi1/2b::eGFP neurons, wesliced fused hSS-hCS into 250 μm sections and performed whole-cellvoltage clamp recordings of synaptic responses. To reliably distinguishbetween excitatory postsynaptic currents (EPSCs, downward deflecting)and IPSCs (upward deflecting), we used a low Cl— solution in the patchpipette and held the cells at ˜50 mV. We found that Dlxi1/2b::eGFP thatmigrated into hCS display both EPSCs and IPSCs (FIG. 4j ). Moreover,after migration into hCS, these cells primarily receive EPSCs ratherthan IPSCs and their synaptic input increases approximately 3-fold (FIG.4k , P<0.001; FIG. 14d ; electrical properties of patched cells areshown in Table 5). In parallel, glutamatergic neurons from hCS, whichexhibit only EPSCs before fusion, also begin receiving IPSCs and show anoverall increase in synaptic input following the migration ofinterneurons from hSS (FIG. 4l , FIG. 14e , P<0.05). Lastly, in order toassess the functional integration of hCS and hSS neurons into neuralnetworks, we applied electrical stimulation to the hCS side of fusedhSS-hCS to trigger glutamate release from excitatory neurons in thevicinity of the stimulation electrode while simultaneously recordingEPSCs and IPSCs in Dlxi1/2b::eGFP cells that migrated into hCS (FIG. 4m; FIG. 14f ). We found that electrical simulation evoked EPSCs (eEPSCs)immediately following simulation (>5 ms); this was followed by presumeddisynaptic IPSCs (<15 ms) sensitive to gabazine, suggesting the assemblyof a 3D cortical microphysiological system that incorporates bothexcitatory and inhibitory synaptic activity.

TABLE 5 SUPPLEMENTARY TABLE 5 Max Half- RMP* Rheobase ThresholdSpike/Sec Overshoot Width EPSPs/min IPSPs/min n DIxI1/2b::eGFP −61.8 ±3.2 6.1 ± 0.7 −46.8 ± 4.2 3.1 ± 0.6  8.6 ± 1.6 4.2 ± 0.3 2.0 ± 0.7 8.7 ±1.6 11 neurons in hSS DIxi1/2b::eGFP −86.4 ± 4.7 5.6 ± 0.7 −49.3 ± 5.06.8 ± 1.0 15.6 ± 2.1 3.8 ± 0.2 15.5 ± 2.2  3.6 ± 1.0 11 neurons In hCSDIxi1/2b::aGFP −83.4 ± 2.9 8.2 ± 0.9 −42.5 ± 3.2 2.8 ± 0.5 12.2 ± 1.63.9 ± 0.4 0.4 ± 0.2 5.5 ± 1.8 15 neurons In unfused hCS neurons in −61.2± 4.3 6.6 ± 1.1 −41.7 ± 3.9 4.5 ± 1.0 10.8 ± 0.8 5.8 ± 0.7 11.0 ± 1.7 6.8 ± 2.3 9 fused hCS neurons in −59.8 ± 3.9 8.8 ± 1.4 −44.7 ± 4.3 4.2 ±0.7 10.5 ± 1.2 6.2 ± 0.8 8.3 ± 2.0 0.2 ± 0.2 6 unfused *Resting MembranePotential

In this study, we show the generation of a human 3D microphysiologicalsystem (MPS) that includes functionally-integrated excitatoryglutamatergic and GABAergic neurons. This platform has severaladvantages in comparison to previous methods for deriving organoids orcortical interneurons in adherent conditions. First, it involves thedirected differentiation of subdomains of the forebrain thatfunctionally interact in development. In contrast to whole-brainorganoids and organoids resembling broader brain regions, this approachallows for modularity by combining separately patterned spheroids intomulti-region neural 3D cultures. Second, this system captures in vitromore elaborate processes during CNS development, including the saltatorymigration of interneurons towards the cerebral cortex. Using liveimaging of the mid-fetal human forebrain, we demonstrate that thissaltatory interneuron migration is accurately recapitulated with thisassembled 3D platform. Third, by enabling their migration into an activeneural network, interneurons mature and integrate into a synapticallyconnected microphysiological system without the requirement of seedingonto rodent cortical cultures or brain slices. Assembling networks usingthis modular system may facilitate the study of excitation to inhibitioninterplay during cortical development.

We also demonstrate that forebrain subdomains derived from hiPSCs andfused in vitro can be used to identify the transcriptional changesassociated with interneuron migration and to model disease processesthat take place in mid to late human fetal development and are otherwiseinaccessible. We find that cortical interneurons derived from TSsubjects carrying a mutation in the Cav1.2 channel display acell-autonomous migration defect whereby they move more frequently butless efficiently. Moreover, the abnormal migration in TS cultures isrescued by pharmacologically manipulating voltage-gated calciumchannels, further demonstrating the key role of calcium signaling incortical assembly. This aberrant interneuron migration taken togetherwith our previous studies showing defects in cortical excitatoryneurons, suggest the presence of abnormal cortical development andfunction in TS.

Lastly, the specification in vitro of various subdomains of thedeveloping human brain from hPSC and their assembly into 3D cultures,opens the opportunity for studying the interaction of specific neuronalcell types and for generating and probing microphysiological systemsthat includes neural circuits.

Material and Methods

Culture of hiPSCs. The lines of hiPSC used in this study were validatedusing standardized methods as previously shown. Cultures were tested forand maintained Mycoplasma free. A total of 6 control iPSC lines derivedfrom 5 subjects, plus the human embryonic stem cell line H9, and 7 hiPSClines derived from 3 subjects with TS carrying the pG406R mutation wereused for experiments (Table 1). The TS point mutation in exon 8a ofCACNA1C was verified by PCR as previously described. The hiPSC lineH20961 was derived by the Gilad laboratory. Approval for this study wasobtained from the Stanford IRB Panel and informed consent was obtainedfrom all subjects.

Generation from hiPSC of hCS and hSS. Human pluripotent stem cells(hiPSC or hESC) were cultured on inactivated mouse embryonic fibroblastfeeders (EmbryoMax PMEF; Millipore) in hPSC medium containing DMEM/F12(1:1, Life Technologies, 11330), Knockout Serum (20%, Life Technologies,10828), non-essential amino acids (1 mM, Life Technologies, 11140),GlutaMax (1:200, Life Technologies, 35050), β-mercaptoethanol (0.1 mM;Sigma-Aldrich M3148), penicillin and streptomycin (1:100, LifeTechnologies, 15070), and supplemented with FGF2 (10 ng/ml diluted in0.1% BSA; R&D Systems).

The generation of hCS from hiPSC was performed as previously described.To initiate the generation of hCS or hSS, intact hiPSC colonies werelifted from the plates using dispase (0.35 mg/ml) and transferred intoultralow attachment plastic dishes (Corning) in hPSC medium supplementedwith the two SMAD inhibitors dorsomorphin (DM; 5 μM; Sigma) andSB-431542 (SB; 10 μM, Tocris), and the ROCK inhibitor Y-27632 (10 μM;EMD Chemicals). For the first five days, the hPSC medium was changedevery day and supplemented with dorsomorphin and SB-431542. On the sixthday in suspension, neural spheroids were transferred to neural medium(NM) containing Neurobasal-A (Life Technologies, 10888), B-27 supplementwithout vitamin A (Life Technologies, 12587), GlutaMax (LifeTechnologies, 1:100), penicillin and streptomycin (Life Technologies,1:100) and supplement with the growth factors EGF (20 ng/ml; R&DSystems) and FGF2 (20 ng/ml; R&D Systems) until day 24. For thegeneration of hSS, the medium was supplemented with additional smallmolecules during the first 23 days in culture; a schematic showing therecipes is presented in FIG. 8a ). The hSS-IS condition involved theaddition of the Wnt pathway inhibitor IWP-2 (5 μM; Selleckchem) from day4 until day 24, and the SHH pathway agonist SAG (smoothened agonist; 100nM; Selleckchem) from day 12 to day 24. The hSS-ISA condition alsoincluded IWP2 and SAG with the addition of allopregnanolone (AlloP 100nM; Cayman Chemicals) from day 15 to day 23, while the hSS-ISRAcondition included AlloP (100 nM) from day 15-23, and a brief exposure(day 12-15) to retinoic acid (RA 100 nM; Sigma). From day 25 to 42, theNM for both the hCS and hSS conditions, was supplemented with the growthfactors BDNF (20 ng/ml; Peprotech) and NT3 (20 ng/ml; Peprotech) withmedium changes every other day. From day 43 onwards, hCS and hSS weremaintained in unsupplemented NM with medium changes every four to sixdays.

Viral labeling and fusion of neural spheroids. hCS or hSS weretransferred to a 1.5 ml Eppendorf tube containing 300 μl NM with virusand incubated overnight. The next day, neural spheroids were transferredinto fresh NM medium in ultralow attachment plates. Lentivirus(Lenti-Dlxi1/2b::eGFP) was generated by transfecting HEK293T cells withLipofectamine 2000 (Thermo Fisher Scientific) and concentrating thesupernatant with the Lenti-X concentrator (Clontech) 72 hrs later.Adenovirus (AAV-DJ1-hSyn1::mCherry) was generated in the Stanford GeneVector and Virus Core at Stanford.

To fuse the forebrain spheroids, hCS and hSS (˜60 to 90 days of in vitrodifferentiation), which were virally labeled 8-10 days before, weretransferred to a 1.5 ml Eppendorf tube for three days and placed in anincubator. During this time, more than 95% of hCS and hSS fused. ThesehSS-hCS cultures were carefully transferred into 24 well ultralowattachment plates (Corning) using a cut P-1000 pipette tip and mediumchanges were performed very gently every two to three days.

Cryopreservation. hCS were fixed in 4% paraformaldehyde (PFA) and 8%sucrose for 30 min to 2 hrs. They were then washed in PBS, transferredto 15% sucrose solution over night at 4° C. and then to 30% sucrose for48-72 hrs. Subsequently, they were transferred into embedding medium(Tissue-Tek OCT Compound 4583, Sakura Finetek), snap-frozen on dry iceand stored at −80° C. For immunohistochemistry, 10 to 20 μm thicksections were cut using a cryostat (Leica). Human brain tissue was fixedin 4% PFA for 48 hrs, washed in PBS and transferred to 30% sucrose forone week. Sections were then embedded in OCT and 30% sucrose (1:1) andsectioned into 40 μm sections using a Leica cryostat.

Immunohistochemistry. Cryosections were washed with PBS to remove excessOCT and blocked in 10% goat serum (NGS), 0.3% Triton X-100 diluted inPBS for 1 hr at room temperature. The sections were then incubatedovernight at 4° C. with primary antibodies diluted in PBS containing 10%GS and 0.3% Triton X-100. PBS was used to wash off the primaryantibodies and the cryosections were incubated with secondary antibodiesin PBS with 10% NGS and 0.3% Triton X-100 for 1 hr. The followingprimary antibodies were used for immunohistochemistry: anti-NKX2.1(rabbit, 1:200; Santa Cruz: sc-13040), anti-MAP2 (guinea pig, 1:1000;Synaptic Systems: 188004), anti-GABA (rabbit, 1:1000; Sigma: A2052),anti-GAD67 (mouse, 1:1000; Millipore: MAB5406), anti-SST (rat, 1:200;Millipore: MAB354), anti-CR (rabbit, 1:1000; Swant: CR7697), anti-CB(rabbit, 1:1000; Swant: CB38), anti-PV (rabbit, 1:6000; Swant: PV27),anti-PV (mouse 1:1000; Millipore: MAB1572), anti-GFP (chicken, 1:1500;GeneTex: GTX13970), anti-DCX (guinea pig, 1:1000; Millipore: AB2253);anti-TBR1 (rabbit, 1:200; Abcam: AB31940), anti-GFAP (rabbit, 1:1000;DAKO Z0334), anti-CTIP2 (rat, 1:300; Abcam: AB18465), anti-OCT4 (rabbit,1:200, Cell Signaling Technology), anti-SSEA4 (mouse, 1:200, CellSignaling Technology). AlexaFluo Dyes (Life Technologies) were used at1:1000 dilution for amplifying the signal. Nuclei were visualized withHoechst 33258 (Life Technologies). Cryosections were mounted formicroscopy on glass slides using Aquamount (Thermo Scientific) andimaged on a Zeiss M1 Axioscope or Leica TCS SP8 confocal microscope.Images were processed in ImageJ (Fiji).

Dissociation of hCS and hSS. For the enzymatic dissociation of hCS andhSS for culture in monolayer and immunocytochemistry, spheroids wereincubated with Accutase (Innovative Cell Technologies) for 25 min at 37°C., washed with NM and gently triturated using a P-200 pipet. Cells wereplated on poly-ornithine/laminin (Sigma) coated glass coverslips (15 mm;Werner) at a density of ˜1 spheroid per two coverslips in NMsupplemented for BDNF and NT3. To dissociate hCS and hSS for single cellprofiling, we adapted a previously published protocol used for primaryhuman fetal brain tissue 52. Briefly, up to 6 spheroids were choppedusing a #10 blade and then incubated in papain enzyme solution (27.3U/ml; Worthington), EBSS (1×, Sigma), 0.46% Sucrose (Sigma), 26 mMNaHCO3 (Sigma), 0.5 mM EDTA (Sigma) at 37° C. for 70 min in an incubator(5% CO2). The digested spheroids were then washed and carefullytriturated in a trypsin inhibitor solution EBSS, 0.46% Sucrose (Sigma),26 mM NaHCO3 (Sigma), 15-30 mg Trypsin Inhibitor (Sigma). Aftercentrifugation, the pellet was resuspended in 0.2% BSA diluted in PBSand supplemented with Y-27632 (10 μM; EMD Chemicals) and the cells wereused for FACS.

Mouse slice cultures. Whole brains from E14-E18 mouse embryos wereembedded in 4% low-melting point agarose and slices were cut at 250-300μm using a Leica VT1200 vibrotome in complete HBSS (100 ml of 10×HBSSwithout Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30 ml of 1MD-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1 MNaHCO3). Slices with visible forebrain structures were placed inmembrane inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated withPoly-L-orthinine and Laminin (Sigma) overnight. They were cultured in aBasal Medium Eagle (39 mL, Life Technologies, #21010046) supplementedwith 12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mMGlutaMax (Life Technologies) and 5% heat-inactivated horse serum (LifeTechnologies, 26050070). The slices were imaged using a Leica SP8confocal microscope.

Electroporation of mouse slices. Coronal slices of mouse embryonicforebrain at E14 were prepared as described above. Sections weretransferred into tissue culture dishes containing complete HBSS for ˜1hour, after which CAG-Cav1.2 (WT- or TS-CACNA1C) plasmids were focallyco-injected with CAG::GFP at a ratio of 1:0.5 directly into theganglionic eminence through a glass micropipette. Cav1.2 overexpressionconstructs were generated by insertion of PCR-amplified WT- andTS-Cav1.2 coding sequences from dihydropyridine-insensitive Cav1.2constructs47 into pCAGIG (kind gift from C. Cepko through Addgene,plasmid 11159). Slices were then electroporated using two horizontallyoriented platinum electrodes powered by a BTX Square PulseElectroporator, and placed onto cell culture membrane inserts forsubsequent live imaging 48 hrs later as described below.

Human Tissue. Human tissue was obtained under a protocol approved by theResearch Compliance Office at Stanford University. The tissue wasprocessed using an adapted protocol55. Briefly, GW18 or GW20 frontalbrain tissue was embedded in 4% low-melting point agarose in bubbledartificial cerebrospinal fluid (ACSF: 125 mM NaCl, 2.5 mM KCl, 1 mMMgCl2, 2 mM CaCl2, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM D-(+)-Glucose)and either sectioned using a Leica VT1200 Vibratome at 300-500 μm inice-cold, bubbled ACSF, or cut using the sharp end of a gauge-22 needleto obtain 1-2 mm thick sections. The sections were then placed in tissueculture plates containing culture media (66% BME, 25% Hanks, 5% FBS, 1%N-2, 1% penicillin, streptomycin and glutamine; all from Invitrogen) and0.66% D-(+)-Glucose (Sigma) and incubated (37° C., 5% CO2) with theDlxi1/2b::eGFP lentivirus for 30 min to 1 hr. Sections were thentransferred to cell culture membrane inserts (diameter, 13 mm; poresize, 8 μm; Costar) and incubated in culture media at 37° C., 8% O2, 5%CO2 for up to 8 days. Half media changes were performed every other day.After ˜5 days in culture, Dlxi1/2b::eGFP+ cells could be detected andwere subsequently imaged as described below.

Live imaging and analysis of Dlxi1/2b::eGFP+ cell migration. Themigration of Dlxi1/2b::eGFP+ cells was imaged for 8-12 hrs underenvironmentally controlled conditions (37° C., 5% CO2) in intact, fusedhSS-hCS using a confocal microscope with a motorized stage (Leica SP8).Fused hSS-hCS were transferred to a well of a 96-well plate(glass-bottom plates, Corning) in 200 μl of NM. Spheroids were incubatedin the environmentally controlled chamber for 30-60 min prior toimaging. During a given recording session, up to 8 fused hSS-hCS wereimaged at a depth of 50-150 μm depth and a rate of 15-20 min/frame. Forpharmacological manipulation, cells were imaged for 12 hrs to record abaseline. Then, the media was carefully removed and new media with smallmolecules (AMD3100 at 100 nM; nimodipine at 5 μM; or roscovitine at 15μM) was gently added to the well. The field of view was readjusted tocapture the previous region of interest and cells in fused hSS-hCS wereimaged for an additional 12 hrs. For imaging of Dlxi1/2b::eGFP+ cells,E17-E18 slices were placed on inserts and infected with Dlxi1/2b::eGFPlentivirus after 24 hrs. The slices were imaged 2 days later using aLeica SP8 confocal microscope (see above). For measuring the branch tosoma length ratio of human cells on mouse slices, hSSs infected withDlxi1/2b::eGFP lentivirus were dissociated and placed on top of E13-14mouse slices, which were placed on cell culture inserts 8-24 hrs before.The hSS-derived Dlxi1/2b::eGFP+ cells were imaged with the Leica SP8confocal microscope system at least 48 hrs later. The migration of mouseDlxi1/2b::eGFP+ cells or Cav1.2-electroporated cells and the migrationof human fetal Dlxi1/2b::eGFP-infected cells both imaged using the samesetting as described for intact, fused hSS-hCS. Slices were kept on thecell culture inserts during imaging. For quantification of migration ofDlxi1/2b::eGFP+ cells after plating on coverslips, intact hSS wereplated on Poly-ornithine/laminin (Sigma) coated glass coverslips (15 mm;Werner). Cells were imaged 7-10 days after using a confocal microscope(Leica SP8) as described above. ImageJ and the Chemotaxis & MigrationTool (Ibidi) were used for the post-acquisition analysis of cellmobility. The StackReg plugin in ImageJ was used to correct for minordrifts during imaging. To estimate the length of individual saltations,Dlxi1/2b::eGFP cells displaying a swelling of the soma were identified,and distance (in μm) to the new position of the soma followingnucleokinesis was recorded manually. The time necessary for thismovement was used to calculate the speed when mobile. To estimatedirectness of movement, the x and y coordinates of each cell per frameand time were extracted with the Manual Tracking plugin (ImageJ) and theChemotaxis & Migration Tool (Ibidi) was used to calculate theAccumulated (A) and Euclidian (E) distances traveled per cell over time.Path directness was calculated as the E/A ratio. Videos were processedusing ImageJ and Final Cut Pro X.

Fura-2 calcium imaging of hSS or hCS cultures Dissociated hSS (day 62)or hCS (day 123) derived from control and TS lines were cultured onpoly-L-ornithine and laminin (Sigma) coated coverslips for 4-5 days. Thecultures were incubated with 1 μM Fura-2 acetoxymethyl ester (Fura-2AM;Invitrogen) for 25 min at 37° C. in NM medium, washed for 5 min andplaced in a perfusion chamber on the stage of an inverted fluorescencemicroscope (TE2000U; Nikon). Cells were then stimulated with high-KClTyrode's solution (67 mM KCl, 67 mM NaCl2 mM CaCl2, 1 mM MgCl2, 30 mMglucose and 25 mM HEPES, pH 7.4). Imaging was performed at roomtemperature (25° C.) on an epifluorescence microscope equipped with anexcitation filter wheel and an automated stage. Openlab software(PerkinElmer) was used to collect and quantify time-lapse excitationratio images. Fluorescence images were analyzed using the IGOR Prosoftware (WaveMetrics). Residual calcium following high-KCldepolarization was calculated by dividing the plateau calcium level bythe peak calcium elevation ((C−A)/(B−A); FIG. 3b ).

Fluo-4 calcium imaging in intact hSS. Intact hSS at day 43-52 wereincubated with 10 μM Fluo-4 acetoxymethyl ester (Fluo-4AM; Invitrogen)for 30 min in NM media followed by a 15 min wash with NM. A Leica SP8confocal microscope with a resonant scanner was used for imaging.Spontaneous calcium activity was recorded for 10 min (one frame every8-10 s) in one 10 μm z-stack plane. Fluorescence intensity (F) wasexported as mean gray values in ImageJ. Signal decay was controlled bysubtracting the mean fluorescence of the background (Fb). To estimatechanges in intracellular calcium, ΔF was computed as (Fcell−Fb)/F0,where F0 represents the minimum F value per cell across the whole 10 minof recording from which Fb was subtracted. A ΔF>1.2 was defined as aspike.

iDISCO. To optically clear fixed fused spheroids, we adapted the iDISCOprotocol described by Renier et al56. Briefly, after fixation with 4%PFA for 3 hrs, spheroids were dehydrated with a day-long methanol(MetOH) dilution series (20% to 100% MetOH). Next, they were incubatedin 5% H2O2 overnight at 4° C. The following day, they were rehydratedwith a reverse MetOH dilution series and incubated overnight in 0.2%Triton-X, 20% DMSO, 0.3 M Glycine/PBS at 37° C. The spheroids were thenblocked with 0.2% Triton-X, 10% DMSO, 6% goat serum/PBS at 37° C. for 2days, followed by a heparin treatment for 2 hrs (PTwH: 0.2% Tween-20, 10μg/mL Heparin/PBS) to reduce non-specific antibody binding. They werenext incubated with a chicken anti-GFP (1:1500; GeneTex: GTX13970)antibody for 2 days in PTwH with 5% DMSO and 3% goat serum at 37° C.After a day-long wash series with PTwH, a secondary antibody diluted inPTwH, 3% Donkey Serum was added for an additional two days at 37° C.After 2 days of PTwH washes, the spheroids were cleared by a three-steptetrahydrofuran (THF) series (80%, 100%, 100% THF/H2O), a 10 mindichloromethane step, and a short incubation in dichloromethane (DBE).The cleared spheroids were stored and imaged in DBE on a Leica SP8confocal microscope.

Real time quantitative PCR (qPCR). mRNA was isolated using the RNeasyMini Kit and RNase-Free DNase set (Qiagen), and template cDNA wasprepared by reverse transcription using the SuperScript III First-StrandSynthesis SuperMix for qRT-PCR (Life Technologies). Real time qPCR wasperformed using SYBR GREEN (Roche) on a ViiA7 machine (AppliedBiosystems, Life Technologies). Data was processed using the QuantStudioRT-PCR software (Applied Biosystems).

Single cell gene expression (BD Resolve system). To capturetranscriptomic information of hiPSCs-derived hCS and hSS (IS)single-cells, we used the BD™ Resolve system (BD Genomics, Menlo Park,Calif.) as previously reported with modifications. Multiple hCS or hSSat day 105 of differentiation were combined and dissociatedenzymatically into single cells, and processed in one batch. Single cellcapture was achieved by random distribution of a single cell suspensionacross >200,000 microwells via a limited dilution approach. Beads witholigonucleotide barcodes were added to saturation such that a bead waspaired with a cell in a microwell. Cell lysis buffer was added such thatpoly-adenylated RNA molecules hybridized to the beads. Beads werecollected into a single tube for reverse transcription. Upon cDNAsynthesis, each cDNA molecule was tagged on the 5′ end (i.e., 3′ end ofmRNA transcript) with a molecular index and cell label indicating itscell of origin. Whole transcriptome libraries were prepared using the BDResolve single cell whole transcriptome amplification workflow. Briefly,second strand cDNA was synthesized, followed by ligation of adaptor foruniversal amplification. Eighteen cycles of PCR were used to amplify theadaptor ligated cDNA products. Sequencing libraries were prepared usingrandom priming PCR of the whole transcriptome amplification products toenrich the 3′ end of the transcripts linked with the cell label andmolecular indices.

Sequencing libraries were quantified using a High Sensitivity DNA Chip(Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay(Thermo Fisher Scientific). 1.5 pM of the library for each sample wasloaded onto a NextSeq 500 system and sequenced using High Outputsequencing kits (75×2 bp) (IIlumina).

The BD Resolve analysis pipeline is used to process sequencing data(fastq files). Cell labels and molecular indices were identified, andgene identity was determined by alignment against the gencodecomprehensive hg19 reference. A table containing molecule counts pergene per cell was output. 7,663 and 4,983 cells were identified for hCSand hSS, respectively, with an average number of reads of ˜14,800, anaverage of ˜3,710 molecules and ˜1,700 number genes detected per cellwith an average molecular index coverage (i.e. number of times amolecule was sequenced) of ˜2. A total of 34,242 genes were detectedacross all cells. Cells with mitochondrial gene (with gene symbolstarting with MT) content>25%, were discarded retaining 7,126 and 4,712cells for hCS and hSS (IS), respectively. Pseudogenes were removed. Thedistribution of reads per single cell is shown in FIG. 6I. Forvisualization and clustering, the data tables of the two libraries wereconcatenated, and the combined table was further reduced to retain onlythe most variable genes using the method outlined in Macosko et al57,yielding 1,102 genes. t-SNE projection of the data was performed usingdefault parameters19. To determine the set of genes contributing to theseparation of cell clusters, differential gene expression analysis(DEseq) based on negative binomial distribution58 was conducted tocompare gene expression profiles in cells in each cluster versus thosein the rest of the data set. Genes are ranked by smallest P values(expressed in terms of −log 10) and the list of significantlyover-represented genes with −log 10 (p-value)<10 of each cluster isprovided as Table 2. Patterns of expression for the top 25 genes in eachcluster is shown in FIG. 6d-k ).

Single cell RNA-seq (Smart-seq2). For assessing gene expression inDlxi1/2b::eGFP+ cells before and after migration, we used a single-cellRNA-Seq assay adapted from the Smart-seq-2 protocol reported by Picelliet al. In short, hSS and hCS that had been fused for ˜15 days wereseparated with a scalpel blade and dissociated independently asdescribed. Single-cells were isolated by FACS into a 96-well PCR platecontaining 5 μl of lysis buffer containing 0.04% Triton X-100 (10%,Sigma BioUltra), 0.1 μl recombinant RNase inhibitor (TaKaRa), 1 μlOligo-dT30VN (10 μM), 1 μl of 10 mM dNTP mix (Fermentas) andnuclease-free H2O for a final volume of 5 μl. A known number of internalRNA control (ERCC) was added to the lysis master mix to estimate thetechnical variability between the wells of the same plate and betweenplates. Reverse transcription and PCR amplification were performed usingthe parameters described by Picelli et al. The quality of the cDNAlibrary was checked using a High-Sensitivity DNA chip (AgilentBioanalyzer). Libraries were prepared using the Nextera XT library prepkit (96 index primers, Illumina). Because the Nextera XT kit is verysensitive to the concentration of cDNA, we screened pre-amplified cDNAlibraries from all plates using the Qubit dsDNA HS Assay kit and used125 pg cDNA from each positive well to further process the tagmentationand indexing. We used 12 additional PCR cycles to further enrich forpre-amplified tagmented DNA. The quality of the tagmented library waschecked using the High-Sensitivity Bioanalyzer chip. The final pooledlibrary was diluted to 2 nM using the elution buffer (Qiagen), and 10 pMwas loaded on an Illumina HiSeq 2500 instrument for sequencing.Libraries were sequenced to obtain 50 bp single end reads (TruSeq Rapidkit, Illumina) with 8 additional cycles for indexing. On average, weobtained 2 million pass filter reads per single cell (FIG. 11c ). Weconsidered a gene expressed if there were at least 10 reads detected forthat gene. Cells that expressed more than 1,000 genes and <10%mitochondrial RNAs were kept for analysis. To avoid bias during FACSfrom RNA contamination from the glutamatergic neurons on the hCS side ofthe fused hSS-hCS, we analyzed STMN2+ cells that did not expressedSLC17A6 or SLC17A7. To control for technical noise, we used aquantitative statistical analysis60 to detect biological variable genesand used them for further analysis. To cluster and visualize the cells,we used the tSNE method in the computational software package Seurat.

Array tomography (AT). AT was used to collect high-resolution images ofsynapses within neural spheroids using previously published protocols.Briefly, fused hSS-hCS were fixed in 4% paraformaldehyde, 2.5% sucrosein phosphate buffered saline. To preserve GFP fluorescence, the tissuewas dehydrated up to 70% alcohol only, with processing through 50%ethanol, 70% ethanol, 1:3 70% ethanol:LR White Resin (LRW, medium grade,SPI supplies), and LRW overnight before embedding in LRW. The embeddedtissue was sectioned into ribbons of 70 nm thick sections (˜30sections/ribbon) and each ribbon was immunostained in 2-3 rounds ofstaining with the antibodies eluted after each round. The followingprimary antibodies were used for immunostaining: anti-GFP (chicken,1:200; Genetex: 13970, 1:200), anti-SYN1 (rabbit, Cell Signalling:5297S, 1:500), anti-PSD95 (rabbit, Cell Signalling: 3450S), anti-VGUT1(guinea pig, 1:5000; Millipore: AB5905), anti-Gephyrin (mouse, 1:100; BDBiosciences: 612632), anti-VGAT (guinea pig, 1:200; Synaptic Systems131004), anti-VGAT (mouse, 1:200; Synaptic Systems: 131011), anti-GFAP(chicken, 1:300; Ayes), anti-MAP2 (guinea pig, 1:1000; Synaptic Systems:188004). Sections were visualized on a Zeiss Axio Imager.Z1 uprightfluorescence microscope using AxioVision software (rel 4.7, Zeiss).Images were processed and registered using FIJI/ImageJ with standard andcustom plugins.

Electrophysiology. Sections of hCS, hSS (day 96-141) or fused hSS-hCS(29-53 daf) for physiological recordings were obtained using an approachwe previously described16. Briefly, spheroids were incubated inbicarbonate buffered artificial cerebrospinal fluid (ACSF) at 23° C. andequilibrated with a mixture of 95% O2 and 5% CO2. The ACSF solutioncontained: 126 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 1.25 mMNaH2PO4, 1 mM MgSO4, and 2 mM CaCl2. Slicing was performed using a LeicaVT1200 vibratome. Immediately after sectioning, slices were moved to acirculation chamber containing ACSF at 32° C.

For patch-clamp recording, cells were identified by the presence of afluorescent reporter using an upright Axoscop II microscope (Zeiss).Recording electrodes of borosilicate glass had a resistance of 4-6 MΩwhen filled with internal solution. A low Cl— internal solution was usedto distinguish between EPSCs and IPSCs containing: 145 mM K+ gluconate,0.1 mM CaCl2, 2.5 mM MgCl2, 10 mM HEPES, 0.2 mM EGTA, 4 mM Na+phosphocreatine. Cl— reversal was calculated to be at −91 mV accordingto the Nernst equation. A high Cl— internal solution was used to measureEPSCs in a subset of unfused hSS containing: 135 mM CsCl, 10 mM HEPES,10 mM EGTA, 3 mM MgATP, 0.3 mM GTP. The Cl— reversal potential wascalculated to be 0 mV according to the Nernst equation. IPSCs wereblocked by application of the GABAA receptor antagonist gabazine (10 μM,Abcam), which was added to superf used ACSF. EPSCs were blocked byapplication of the glutamate receptor antagonist kynurenic acid (1 mM,Abcam), which was added to superf used ACSF. Electrical simulation wasdelivered using a bipolar tungsten electrode (FHS) placed 200-400 μMaway from a recorded neuron. Stimulations were delivered to slices for0.1 ms at 300 μV and separated by at least 10 s. Inward EPSCs andoutward IPSPs were recorded by filling the patch pipette with a lowchloride internal solution (ECl—=−90 mV) and holding the cell at −40 mV,which is the midpoint between ECl— and EK+/Na+. Data were collectedusing a 1550A digitizer (Molecular Devices), a 700B patch-clampamplifier (Molecular Devices) and acquired with the pClamp 10.6 software(Molecular Devices). Recordings were filtered at 10 kHz. Synapticrecordings were analyzed using custom software developed by J.R.H.(Wdetecta). Action potentials were analyzed using custom MATLAB(MathWorks) programs. IPSCs and EPSCs were detected based on theirdirection and shape. We calculated the first time derivative of thecurrent recording and set a detection threshold that was above the noisefor each trace. Detected responses were then evaluated to confirm thedetection accuracy.

Gene expression accession codes. Gene expression data are available inthe Gene Expression Omnibus under accession codes: GSE93811 (BD™Resolve) and GSE93321 (Smartseq-2).

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

That which is claimed is:
 1. A method for producing an integrated humanforebrain structure comprising human GABAergic interneurons synapticallyintegrated with human glutamatergic neurons in vitro, the methodcomprising: (A) inducing in a pluripotent stem cell suspension culture aneural fate by culturing pluripotent stem cells in medium comprising aneffective dose of an inhibitor of bone morphogenetic protein (BMP) andan inhibitor of TGFβ to derive spheroids of neural progenitor cells; (B)differentiating neural progenitor cells in a spheroid of step A intocortical spheroids (hCS) by the steps comprising: (i) culturing thespheroid in neural medium comprising an effective dose of fibroblastgrowth factor 2 (FGF2) and epidermal growth factor (EGF) for a period offrom 1 to 4 weeks; (ii) moving the spheroid to medium comprising aneffective dose of brain-derived neurotrophic factor (BDNF) andneurotrophin-3 (NT3) for a period of from 4 to 7 weeks to generate hCS;(C) differentiating neural progenitor cells in a spheroid of step A tosubpallial spheroids (hSS) by the steps comprising: (i) culturing aspheroid of step A in the presence of medium comprising an effectivedose of a Wnt inhibitor and Agonist of Smoothened for a period of from 7to 24 days; (ii) culturing the spheroid in neural medium comprising aneffective dose of fibroblast growth factor 2 (FGF2) and epidermal growthfactor (EGF) for a period of from 1 to 4 weeks; (iii) moving thespheroid to medium comprising an effective dose of brain-derivedneurotrophic factor (BDNF) and neurotrophin-3 (NT3) for a period of from4 to 7 weeks to generate hSS; (D) culturing the hCS and hSS in closeproximity for an extended period of time in neural medium to provide anintegrated forebrain structure called an assembled organoid comprisinghuman GABAergic interneurons synaptically integrated with humanglutamatergic neurons.
 2. The method of claim 1, wherein the integratedhuman forebrain structure comprises cells differentiated into one ormore of neurons, glial cells, neuro-epithelial cells, andoligodendrocytes.
 3. The method of claim 1, wherein the pluripotent stemcells or derived neural cells comprise at least one genetic, geneticvariant or genetic mutation associated with a neurologic or psychiatricdisorder.
 4. The method of claim 1, wherein the pluripotent stem cellsare induced pluripotent stem cells.
 5. The method of claim 1, whereinthe culture medium of step (A) comprises dorsomorphin as the inhibitorof BMP and SB-431542 as the inhibitor of TGFβ, each in a dose effectiveto induce pluripotent stem cells to a neural fate.
 6. The method ofclaim 5, wherein the culture of step (A) is feeder layer free.
 7. Themethod of claim 1, wherein the medium of step (C)(i) further comprisesone or both of allopregnanolone and retinoic acid at a dose of from 10nM to 10 μM.
 8. The method of claim 1, wherein the medium of step (A)comprises a selective Rho-associated kinase (ROCK) inhibitor frominitiation of culture for up to 48 hours.
 9. The method of claim 1,wherein in step (D) the hCS and hSS are combined at a ratio of from 1:3to 3:1.